Mycotaxon

YCOTAXON

THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE

VOLUME 134 (4) OCTOBER-DECEMBER 2019

Rhomboidia wuliangshanensis gen. & sp. nov.
(Xu & al.— Fie. 2, p. 657)

ISSN (PRINT) 0093-4666 https://doi.org/10.5248/134-4 ISSN (ONLINE) 2154-8889
MYXNAE 134(4): 591-740 (2019)

EDITORIAL ADVISORY BOARD

KAREN HANSEN (2014-2021), Chair
Stockholm, Sweden

BRANDON MATHENY (2013-2020), Past Chair

Knoxville, Tennessee, U.S.A.

ELSE VELLINGA (2019-2022)
Oakland, California, U.S.A.

XINLI WEI (2019-2023)

Beijing, China

ToDD OSMUNDSON (2019-2024)
La Crosse, Wisconsin, U.S.A.

ELAINE MALosso (2019-2025)
Recife, Brazil

ISSN 0093-4666 (PRINT)
ISSN 2154-8889 (ONLINE)

MYCOTAXON

THE INTERNATIONAL JOURNAL OF FUNGAL TAXONOMY & NOMENCLATURE

OCTOBER-DECEMBER 2019

VOLUME 134 (4)

http://dx.doi.org/10.5248/134-4

EDITOR-IN-CHIEF

LORELEI L. NORVELL
editor@mycotaxon.com

Pacific Northwest Mycology Service
6720 NW Skyline Boulevard
Portland, Oregon 97229-1309 USA

NOMENCLATURE EDITOR

SHAUN R. PENNYCOOK
PennycookS@LandcareResearch.co.nz

Manaaki Whenua Landcare Research
Auckland, New Zealand

MyYcoTAxONn, LTD. © 2019

www.mycotaxon.com &
www.ingentaconnect.com/content/mtax/mt

P.O. BOX 264, ITHACA, NY 14581-0264, USA

Iv ... MYCOTAXON 134(4)

MYCOTAXON

VOLUME ONE HUNDRED THIRTY-FOUR (4) — TABLE OF CONTENTS

134-4: TABLE OF CONTENTS, NOMENCLATURAL UPDATES, PEERS, EDITORIALS

POVIC WONG Perk ols bcs ng ghs_ a tung Sea efor v athe citer sy 3 read RI Us eee Soret get ae vi
Nomernclatural novelties ty pifications soo 4. cok ey tp eras keke eens vii
COPTICONAG RA es Shox. aSecey gen on eau tats eae Este bod Gallente 8a alee g viii
PP ROMISTTCTSAILOT 3, nyc. cess Ses aegy $e stg ents Sea he trv Aes gw cate ce ahh ob ogo eee ix
ZOLO Ss WUMISSIOR DPYOCEAURE? 3: Pe tons Fry Sone Py Ra ver oAd oy Dey ly Yo Sha xi

RESEARCH ARTICLES

Urocystis cumminsii sp. nov., a smut fungus on Themidaceae
from Arizona KYRYLL G. SAVCHENKO, SYLENA R. HARPER,
Lori M. Carrts, Lisa A. CASTLEBURY 591
Leucoagaricus brunneus sp. nov. from Khyber Pakhtunkhwa, Pakistan
Zi1A ULLAH, SANA JABEEN, MUHAMMAD FAISAL,
Hasis AHMAD, ABDUL Nasir KHALID 601
Dictyostelids from Jilin Province, China 3:
new Cavenderia and Dictyostelium records
Pu Liu, SHUNHANG ZHANG, ZHUANG LI*,
YuE Zou, XUEPING KANG, Yu Li 613
Records of Aureobasidium harposporum, Sarcophoma miribelii,
and Stigmina dothideoides from Turkey
MAKBULE ERDOGDU, MERVE ULUKAPI,
ALI IHSAN KARAYEL, ZEKIYE SULUDERE 619
Bactrodesmium pulcherrimum sp. nov. from Ecuador

FERNANDO ESPINOZA, DAYNET SOSA, LIZETTE SERRANO,
ADELA QUEVEDO, FREDDY MAGDAMA, MARCOS VERA,

SIMON PEREZ-MARTINEZ, ELAINE MALOSSO, RAFAEL F. CASTANEDA-RUIZ 627
Dendrographium multiseptatum sp. nov. from China
L1-Guo Ma, YuE-L1 ZHANG, Bo ZHANG,
Kal Qt, CHANG-SONG LI, JUN-SHAN QI 633
Haematomma pluriseptatum sp. nov. from China
CONGCONG MIAO, RONG TANG,
LINLIN DONG, ZHAOJIE REN, ZUNTIAN ZHAO 637
New records of Didymium inconspicuum, D. karstensii, and
D. rugulosporum from China
CHAOFENG YUAN, SHU LI, WAN WANG, SHUWEI WEI, QI WANG, Yu LI 643

OCTOBER-DECEMBER 2019 ... V

Rhomboidia wuliangshanensis gen. & sp. nov.
from southwestern China
TaI-MIN Xu, XIANG-Fu Liu, Yu-Hur CHEN, CHANG-LIN ZHAO 649
Filsoniana lhasanensis sp. nov. from Tibet, China
XuE-MEI! WEN, HurNISA SHAHIDIN, ABDULLA ABBAS 663
Sarcopodium flocculentum, the correct name for S. macalpinei
SHAUN R. PENNYCOOK & PauL M. Kirk) 677

Pteridicolous ascomycetes from a cloud forest in eastern Mexico
ROSARIO MEDEL-ORTIZ, YAJAIRA BAEZA,
FRANCISCO G. LOREA-HERNANDEZ 681

First sexual morph record of Sarcopodium vanillae
NAPALAI CHAIWAN, SAJEEWA S.N. MAHARACHCHIKUMBURA,
DHANUSHKA N. WANASINGHE, MINGKWAN DOILoM,

RUVISHIKA JAYAWARDENA, KEVIN D. HyDE 707

Notes on rust fungi in China 7. Aecidium caulophylli life cycle
inferred from phylogenetic evidence and renamed as
Puccinia caulophylli comb. nov. JiInG-XIn Jt, ZHUANG LI,
Yu Li, MAKOTO KAKISHIMA 719

Exserticlava aquatica sp. nov., a microfungus
from the Brazilian Amazon LUANA TEIXEIRA DO CARMO,
D10GO CARELI DOS SANTOS, CAROLINA RIBEIRO SILVA,
SHEILA MIRANDA LEAO FERREIRA, THAMARA ARAO FELETTI,

Luis FERNANDO PASCHOLATI GuSMAO 731

MycoBioTa (FUNGA) NEW TO THE MYCOTAXON WEBSITE

Ascomycota (lichenized and non-lichenized) on Syagrus coronata
in the Caatinga biome: new and interesting records
for Brazil and South America (suMMARyY)
Mata4ra A.L. Dos SANTOS, NILO G. S. FORTEs,
TAssio E. FE. Sitva, NaApja S. VITORIA 737
Checklist of Bolivian Agaricales. 1:
Species with dark and pink spore prints (suMMaRy)
E. MELGAREJO-ESTRADA, M.E. SUAREZ,
D. RocaBADO, O. MAILLARD, B.E. LECHNER 739

vi ... MYCOTAXON 134(4)

REVIEWERS — VOLUME ONE HUNDRED THIRTY-FOUR (4)

The Editors express their appreciation to the following individuals who have,

prior to acceptance for publication, reviewed one or more of the papers

prepared for this issue.

M. Catherine Aime
D. Jayarama Bhat

Lu-Sen Bian

Marcela Eugenia da Silva Caceres

Rafael F. Castahteda-Ruiz
Vagner G. Cortez
Cvetomir M. Denchev
Shouyu Guo

Tom Hsiang

Shah Hussain

Sevda Kirbag

Klaus Kalb

Paul M. Kirk

John C. Landolt
De-Wei Li

José G. Marmolejo
Tom May

Eric H.C. McKenzie

Josiane Santana Monteiro
Karen K. Nakasone
Nicolas Niveiro
Lorelei L. Norvell
Cumali Ozaslan
Shaun R. Pennycook
Marcin Piatek

Meike Piepenbring
Luis Quijada

Scott Redhead
Andrea Irene Romero

Michelline Lins Silvério

Adna Cristina Barbosa de Sousa

Steven L. Stephenson
Jan Vondrak

Xinli Wei

Ze-Fen Yu

Changlin Zhao

OcCTOBER-DECEMBER 2019...

NOMENCLATURAL NOVELTIES AND TYPIFICATIONS

PROPOSED IN MYCOTAXON 134(4)

Bactrodesmium pulcherrimum R.F. Castaneda, F. Espinoza & D. Sosa
[MB 830569], p. 629

Dendrographium multiseptatum L.G. Ma & J.S. Qi
[MB 832847], p. 634

Exserticlava aquatica L.T. Carmo, C.R. Silva, Careli, S.M. Leao, Feletti
& Gusmao [MB 831391], p. 732

Filsoniana lhasanensis X.M. Wen, Shahidin & A. Abbas
[FN 570592], p. 669

Haematomma pluriseptatum R. Tang
[MB 830618], p. 638

Leucoagaricus brunneus Z. Ullah, Jabeen & Khalid
[MB 827985], p. 603

Puccinia caulophylli (Kom.) Jing X. Ji & Kakish.
[MB 830631; epitypified: MBT 386779], p. 726

Rhomboidia C.L. Zhao
[MB 833318], p.654

Rhomboidia wuliangshanensis C.L. Zhao
[MB 833320], p. 656

Urocystis cumminsii Savchenko, Carris & Castl.
[MB 830145], p. 595

VII

vill ... MyCOTAXON 134(4)

CORRIGENDA

VOLUME 134-1

p.174, ACKNOWLEDGMENTS
FoR: The author would like to thank Prof. Dr. Ertugrul Sesli, Prof. Dr. Ibrahim
Tirkekul, and Dr. Shaun Pennycook for their helpful comments and careful

review.

READ: The author would like to thank Yiiziincii Yil University, Coordination of
Scientific Research Projects for financial support (2010-FED-B031 and FYL-
2016-5213) and Prof. Dr. Ertugrul Sesli, Prof. Dr. Ibrahim Tiirkekul, and Dr.
Shaun Pennycook for their helpful comments and careful review.

MYCOTAXON 134-3

p. vii, line 21 FoR: Marthamyces culmigenus (Ellis & Everh.) P.R. Johnst.
READ: Marthamyces culmigenus (Ellis & Langl.) P.R. Johnst.

p.496, lines 3-5
FOR: Marthamyces culmigenus (Ellis & Everh.) P.R. Johnst., comb. nov.
IF 556322
= Naemacyclus culmigenus Ellis & Everh..,
Proc. Acad. Nat. Sci. Philadelphia 45: 151, 1893.

READ: Marthamyces culmigenus (Ellis & Langl.) P.R. Johnst., comb. nov.
IF 556322
= Naemacyclus culmigenus Ellis & Langl., in Ellis & Everhart,
Proc. Acad. Nat. Sci. Philadelphia 45: 151, 1893.

[Semi-bold fonts used above to flag corrected terms. ]

CORRIGENDA IN CURRENT ISSUE (134-4)
Cited below are mistakes or oversights present in approved input files not
detected by authors until after PDF conversion.

p. 613: Pu Liu and fellow coauthors wish to acknowledge the contributions made to
“Dictyostelids from Jilin Province, China 3: new Cavenderia and Dictyostelium
records” (MycoTaxon 134: 613-618) by Prof. Zhuang Li (Shandong Provincial
Key Laboratory for Biology of Vegetable Diseases and Insect Pests, College of Plant
Protection, Shandong Agricultural University, Tai’an 271018, China). The author
sequence originally intended should read “Pu Liu, Shunhang Zhang, Zhuang Li,
Yue Zou, Xueping Kang, Yu Li.”

p-669, line 26 FoR: MK43983
READ: MK439830

OCTOBER-DECEMBER 2019 ... IX

FROM THE EDITOR-IN-CHIEF

DEADLINES, MYCOTAXON & NOMENCLATURE— The most onerous task your esteemed
Editor-in-Chief faces is bringing the year-end issue in ‘on time’ A glance at our
publication history suggests that during her tenure, she has failed miserably. Since
2004, she has met only three of her sixteen December deadlines: New Year's Eve
2004, Boxing Day 2007, 30 December 2009. Given this deplorable 37% success rate,
it is no surprise that, once again, an October-December MycotTaxon will appear
in January. (It could be worse: after adopting electronic submission and new software,
we released the last 2005 volume on 11 May 2006!)

Reasons/excuses for delays are myriad: time-consuming nomenclatural revision
and editorial repair of sloppily prepared submissions (on the misplaced notion that
it is quicker for us to do authors’ work for them), outside research commitments,
severe health problems, uncontrollable press delays. Nonetheless, this year we were
convinced that MycoTaxon 134(4) would be ready for delivery well before 2020.

What we did NoT anticipate was the small number of research papers submitted.
With several authors not sending us their final papers after their 2019 nomenclatural
review, we waited until December 27 before deciding to release the issue with
only 140 pages (the smallest issue EVER since 1974) rather than waiting for final
submissions that have yet to appear. Frustrating, because removal of required page
charges produced a flood of 138 accessions during 2019.

What makes the year-end release date so important to MycoTaxon? With
nomenclatural priority date-based, the date of publication is dictated by the actual
release date and not what is displayed on the cover. That means that taxonomic
names published in periodicals must cite both the actual date of publication and
the ‘nominal’ date printed in the issue, e.g., following the order, Haematomma
pluriseptatum R. Tang, Mycotaxon 134: 638 (2020) but indexed as “Miao, C,
Tang R, Dong L, Ren Z, Zhao Z. 2020 (‘2019’). Haematomma pluriseptatum sp.
nov. from China. Mycotaxon 134: 637-641. https://doi.org/10.5248/134.637 ”
Much less confusing and infinitely more restful to have only ONE date to cite!

MyYcOTAXON 134(4) presents 15 papers by 81 authors (representing 15 countries) as
revised by 34 expert reviewers and the editors.

The 2019 October-December MycoTaxon proposes one new genus (Rhomboidia
from China) and eight new species representing Bactrodesmium from ECUADOR;
Dendrographium, Filsoniana, Haematomma, and Rhomboidia from CHINA;
Exserticlava from BraziL; Leucoagaricus from PAKISTAN; and Urocystis from the
U.S.A. We also offer a new combination in Puccinia and epitypification for Puccinia
caulophylli.

New species range extensions are reported for [ascomycetes] Aureobasidium,
Sarcophoma, Stigmina in TURKEY and new records and hosts of fern-associated ascos
in eastern MExico and [myxomycetes] Cavenderia, Dictyostelium, and Didymium
in CHINA.

x ... MYCOTAXON 134(4)

Two papers on Sarcopodium [1] explain why S. flocculentum is the correct name
for S. macalpinei and [2] discuss the first sexual morph recorded for S. vanillae in
Thailand. Another paper treats the full life cycle for the rust Puccinia (= Aecidium)
caulophylli.

Our small year-end issue closes with the announcements of two mycobiota
[recently posted on www.mycotaxon.com] covering [1] new records of ascomycetes
on Syagrus coronata in Brazil's Caatinga biome and [2] dark- and pink-spored agarics
in Bolivia.

Wishing us all health, happiness, illumination, and PEACE in 2020,

Lorelei L. Norvell (Editor-in-Chief)
8 January 2020

PUBLICATION DATE FOR VOLUME ONE HUNDRED THIRTY-FOUR (3)

MYCOTAXON for JULY-SEPTEMBER 2019 (1—-xIv + 423-590)
was issued on October 2, 2019

OCTOBER-DECEMBER 2019 ... XI

2020 MYCOTAXON SUBMISSION PROCEDURE

Prospective MycoTaxon authors should download the MycotTaxon 2020 guide,
review & submission forms, and MycoTaxon sample manuscript by clicking the ‘file
download page’ link on our INSTRUCTIONS TO AUTHORS page before preparing their
manuscript. This page briefly summarizes our “4-step’ submission process.

1—PEER REVIEW: Authors first contact peer reviewers (two for journal papers;
three for mycobiota/fungae) before sending them formatted text & illustration
files and the appropriate 2020 MycoTaxon journal or mycota reviewer comment
form. Experts return revisions & comments to BoTH the Editor-in-Chief
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Place first author surname + genus + ‘MycoTtaxon’ on the subject line, and
(required) attach a completed sUBMISSION FORM. The Nomenclature Editor will
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3—FINAL SUBMISSION: All coauthors thoroughly revise and proof-read files
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the message and all files, which include a (i) revised 2020 submission form, all (ii)
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new name or typification. The Editor-in-Chief acknowledges submissions within
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sending a follow-up query (without attachments).

4—FINAL EDITORIAL REVIEW & PUBLICATION: The Editor-in-Chief conducts a
final grammatical and scientific review and returns her editorial revisions to all
expert reviewers and coauthors for final author approval. Author-approved files
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The PDF proof and bibliographic & nomenclatural index entries are sent to all
coauthors for final inspection. After PDF processing, the Editor-in-Chief corrects
ONLY PDF editorial/conversion and index entry errors; corrections of all other
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The Mycotaxon journal publishes four quarterly issues per year. Both open access
and subscription articles are offered.

MYCOTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 591-599
https://doi.org/10.5248/134.591

Urocystis cumminsii sp. nov., a smut fungus
on Themidaceae from Arizona

KYRYLL G. SAVCHENKO"™*, SYLENA R. HARPER’,
Lori M. Carris’, Lisa A. CASTLEBURY?

Department of Biological Sciences, Butler University, Indianapolis, IN 46208
? Department of Plant Pathology, Washington State University, Pullman, WA 99164, USA

> USDA-ARS, Mycology and Nematology Genetic Diversity and Biology Laboratory,
10300 Baltimore Ave, Beltsville, MD 20705, USA

“ CORRESPONDENCE TO: ksavchen@butler.edu

ABSTRACT—The morphology and phylogenetic relationships of a species of Urocystis on
Dichelostemma capitatum (Themidaceae, Asparagales) collected in the Tucson Mountains in
Arizona, United States, were studied using microscopy and ITS rDNA sequence analyses. This
is a first record for smut fungi on hosts from Themidaceae. Molecular phylogenetic analyses
based on ITS sequence data revealed its basal position in relation to species on Poaceae. As
a result, the smut in leaves of Dichelostemma capitatum is described and illustrated here as a
new species, Urocystis cumminsii.

Key worps—plant pathogens, taxonomy Urocystidales

Introduction

Urocystis Rabenh. ex Fuckel contains more than 170 species of plant
pathogenic smut fungi, found all over the world but more common in
temperate areas of both hemispheres (Vanky 2011b). More than 60% of
Urocystis species are found on monocotyledons, with Poaceae serving as a
major monocotyledonous host family, followed by Juncaceae, Hypoxidaceae,
Convallariaceae, Amaryllidaceae, and Hyacinthaceae (Vanky 2011b). The
intra-level phylogenetic relationships of Urocystis species have never been
analysed and thus far only the graminicolous species from triticoid hosts

592 ... Savchenko & al.

have been included in comprehensive phylogenetic analyses (Savchenko
& al. 2017). Most of the Urocystis species described recently—e.g.,
U. achnatheri L. Guo, U. anemones-narcissiflorae Vanky, U. arxanensis
L. Guo, U. beckwithiae Vanky, U. circaeasteri Vanky, U. dunhuangensis
S.H. He & L. Guo, U. glabella Vanky & R. G. Shivas, U. helanensis L. Guo,
U. koeleriae L. Guo, U. phalaridis Vanky, U. puccinelliae L. Guo & H.C.
Zhang, U. rostrariae Piatek, U. sinensis L. Guo, U. skirgielloae Piatek,
U. wangii L. Guo, and U. xilinhotensis L. Guo & H.C. Zhang—were
supported only by morphological and host-specialization data (Guo 2002,
2005, 2006; Guo & Zhang 2004, 2005; He & Guo 2007; Piatek 2006a,
2006b; Vanky 2004, 2005, 2011la; Vanky & Abbasi 2011). The integration
of molecular phylogenetic analyses, host plant taxonomy, and morphology
provides a natural classification for various smut genera (Bauer & al. 2008;
Castlebury & al. 2005; Kruse & al. 2018; Lutz & al. 2008; McTaggart & al.
2012; Savchenko & al. 2013, 2015; Vanky & Lutz 2007), justifying the need
of a phylogenetic study of the genus.

During a survey of Urocystis species diversity in the United States,
we examined a specimen identified as Urocystis sp. on Dichelostemma
capitatum (Benth.) Alph. Wood (Themidaceae) from Arizona in the WSP
herbarium. Previously, no Urocystis species had been recorded on hosts
from this family. The present study aimed to resolve the specific status of
the smut on D. capitatum through morphological analysis and determine
its phylogenetic affinities within Urocystis.

TABLE 1. GenBank sequences used in this study.

SPECIES GENBANK NO. REFERENCE
Antherospora scillae EF653983 Bauer & al. 2008

A. vaillantii EF653988 Bauer & al. 2008
Urocystis bolivarii KX057771 Savchenko & al. 2017
U. colchici DQ839596 Matheny & al. 2006
U. cumminsii MK575496 This study

U. eranthidis JN367299 Kellner & al. 2011

U. fischeri KF668284 Smith & Lutz 2013

U. occulta KX057774, Savchenko & al. 2017
U. trillii HQ239361 Henricot 2010

U. tritici KX057782 Savchenko & al. 2017
Ustilago hordei AY345003 Stoll & al. 2003
Vankya heufleri EF667965 Bauer & al. 2008

V. ornithogali EF635910 Bauer & al. 2008

Urocystis cumminsii sp. nov. (United States) ... 593

Materials & methods

The herbarium specimen is deposited in Washington State University
Mycological Herbarium, Pullman, WA, United States (WSP).

Sorus and spore characteristics were studied using dried herbarium material.
Specimens were examined by light microscopy (LM). Pictures of sori were taken
with a Canon Power Shot G10 camera. For LM, spores were mounted in 90% lactic
acid on a microscope slide, gently heated to boiling point to eliminate air bubbles,
and then examined under a Carl Zeiss Axiostar™ light microscope at 1000x
magnification and photographed with a Canon Power Shot G10 camera. At least 50
spore balls were measured, and the variation is presented as a range with extreme
values given in parentheses. Means and standard deviations (SD) are provided after
the spore size ranges.

For SEM studies, spore balls were attached to metal stubs by double-sided
adhesive tape and coated with gold. Spore surface ornamentation was observed at
15 kV and photographed with a JEOL JSM-6700F scanning electron microscope
with a working distance of c. 12-13 mm.

Sequences from other species of Urocystis and related genera were obtained
from GenBank (TABLE 1). Genomic DNA was isolated from spore balls removed
from the herbarium specimen that had been lysed in 1.5 mL tubes for 1 min using
FastPrep°24. Tubes were incubated in a water bath for 5 hours at 55 °C, and DNA
extracted using DNeasy Plant Mini Kit (QIAGEN) following the manufacturer’s
instructions.

DNA was amplified in 20 ul aliquots on an Applied Biosystems® GeneAmp 9700
thermal cycler using ITS1 as the forward primer and ITS4 as the reverse primer
(White & al. 1990).

Standard cycling parameters with an annealing temperature of 57 °C were used
for amplification. PCR products were purified with USB ExoSAP-IT according
to the manufacturer’s instructions, amplified with the forward and reverse PCR
primers with the BigDye® Terminator v3.1 Cycle Sequencing Kit, and sequenced on
an ABI PRISM? 3100 Genetic Analyzer.

Consensus sequences were assembled, aligned, and edited with Geneious 7.1.8
for MacOS and with MAFFT 6.853 (Katoh & al. 2002, Katoh & Toh 2008) using the
L-INS-i option. Maximum Likelihood (ML) was implemented as a search criterion
in RAxML (Stamatakis 2014). GTR+I+G was specified as the evolution model in
MrModeltest (Nylander & al. 2004). The RAxML analyses were run with a rapid
Bootstrap analysis (command -fa) using a random starting tree and 1000 maximum
likelihood bootstrap replicates. A Markov Chain Monte Carlo (MCMC) search in
a Bayesian analysis (BA) was conducted with MrBayes (Ronquist & Huelsenbeck
2003). Four runs were implemented for 5 million generations. The cold chain was
heated to a temperature of 0.25°C. Substitution model parameters were sampled
every 500 generations and trees were saved every 1000 generations. Convergence
of the Bayesian analysis was confirmed using AWTY (Nylander & al. 2008) and a

594 ... Savchenko & al.

burn-in of 18,000 generations was calculated. The ML and Bayesian analyses were
run three times to test accuracy. The tree was rooted using Ustilago hordei (Pers.)
Lagerh.

Results

The ITS alignment of 13 sequences (including the outgroup Ustilago hordei)
comprised 643 characters including gaps. The different BA and ML analytical
runs yielded consistent topologies in respect to well-supported branches
(a posteriori probability >90% in most cases). The consensus tree of one run of
Bayesian phylogenetic analyses is presented in Fic. 1. The Urocystis sequences
fell into two major clades. ‘The first clade comprised species from Poaceae and
Themidaceae, and the second clade included species from Cyperaceae, Liliaceae
s.l., and Ranunculaceae. The two species from the genus Vankya Ershad
clustered together with the second Urocystis clade.

Ustilago hordei

Antherospora scillae
Anth Jo)
Antherospora vaillantii a IE Ore
Urocystis cumminsii sp. nov.
Urocystis bolivarii
Urocystis 1

Urocystis occulta Oe 7 is,
Urocystis tritici
Urocystis eranthidis
Vankya heufleri
Vankya ornithogali

Urocystis 2
Urocystis colchici rae = 2

Urocystis trillii

Urocystis fischeri

Fic. 1. Bayesian inference of phylogenetic relationships resulting from the analysis of ITS
nucleotide sequence data. Numbers on branches are estimates for PPs from Bayesian inference
(only probabilities >0.8 are shown).

Urocystis cumminsii sp. nov. (United States) ... 595

Fic. 2. Urocystis cumminsii (WSP 68198). A. sori in leaves of Dichelostemma capitatum; B. spore
balls seen by SEM; C, D. spore balls seen by LM. Scale bars: A = 2 mm, B = 5 um, C, D = 20 um.

Taxonomy

Urocystis cumminsii K.G. Savchenko, Carris & Castl., sp. nov. FIG. 2
MB 830145

Differs from Urocystis camassiae by its lighter colored yellowish-brown spores with
thicker walls and its smaller thinner walled sterile cells, and by its host specialization
on Themidaceae.

Type: USA. Arizona, King’s Canyon, 16 km west of Tucson, on Dichelostemma capitatum
(as D. pulchellum), 30.03.1981, leg. G.B. Cummins (Holotype, WSP 68198; GenBank
MK575496).

EryMoLocy: Named after George B. Cummins (1904-2007), an eminent American
mycologist, who collected the holotype specimen.

596 ... Savchenko & al.

Sor! in leaves as slightly elevated, pustular, elongate areas of various size and
shape, sometimes confluent, visible on both sides of the leaf, initially lead-
colored and covered by the epidermis which ruptures exposing the powdery,
black mass of spore balls. SPORE BALLS globose, subglobose, ovoid to irregular,
20-45 um diam., composed of 1-6 (mostly 3) spores and more or less complete
investing layer of sterile cells. Spores subglobose, ovoid, irregularly oblong to
elongated, 11-15 x 12-18 um diam. [mean + SD, 13.3 + 2.6 x 15 + 2.9 um],
medium yellowish brown, wall 1-1.5 um thick, smooth. STERILE CELLS
subglobose, elongated, ovoid, 5-7 x 5-11(-13) um, pale yellow, to almost
hyaline, with smooth, 1 um thick wall.

Discussion

Dichelostemma Kunth is a North American genus of wild hyacinths, closely
related to Brodiaea Sm., from the family Themidaceae. Plants from this family
are native to Central America and western North America, from British
Columbia to Guatemala (Pires & Sytsma 2002, Stevens 2018). No members of
Themidaceae were previously known to be parasitized by smut fungi (Vanky
2011b). Our molecular phylogenetic analyses and morphological data have
helped resolve the systematic position of Urocystis on D. capitatum.

The only possible close relative to U. cumminsii might be another native
North American species, U. camassiae Vanky, found on Camassia Lindl.
(Agavaceae; Fay & Chase 1996, Pires & Sytsma 2002). However, U. camassiae
is distinguished by its darker colored reddish-brown spores with thinner spore
walls (0.5-1 um) and its larger (5-17 um) sterile cells with thicker walls (1-2
(-3) um; Vanky 1994).

ITS phylogenetic analysis infers that U. cumminsii is sister to the clade of
Urocystis species on grasses and separate from species found on hosts from
families more closely related to Themidaceae, such as U. colchici (Schltdl.)
Rabenh. ex A.A. Fisch. Waldh. and U. trillii H.S. Jacks., indicating multiple
inter-family host jumps during the evolution of Urocystis species, similar to
those in the closely related genus Thecaphora Fingerh. (Vasighzadeh & al.
2014). Interestingly, Vankya heufleri (Fuckel) Ershad and V. ornithogali (J.C.
Schmidt & Kunze) Ershad also clustered within the Urocystis lineage (Fic. 1).

Most sequences of Urocystis in GenBank are derived from the LSU region.
Unfortunately, our preliminary analysis showed that LSU is not informative for
Urocystis phylogenetics. Hence, we based our current phylogeny on ITS data,
utilizing the limited number of Urocystis ITS sequences available in GenBank.
Additional sequencing is needed to resolve the taxonomy and evolutionary

Urocystis cumminsii sp. nov. (United States) ... 597

relationships within Urocystis, as well as the phylogenetic affiliation of closely
related genera, such as Vankya.

Urocystis cumminsii expands the occurrence of Urocystis species to the
host family Themidaceae. Future combined molecular phylogenetic and
morphological analyses may reveal higher diversity among Urocystis species in
North America.

Acknowledgments

The authors are grateful to Mary Catherine Aime (Purdue University, Lafayette IN,
US.A.) and Marcin Piatek (W. Szafer Institute of Botany, Polish Academy of Sciences,
Krakow) for peer reviewing the manuscript and Shaun Pennycook for his valuable
comments. Funding for this work was provided by USDA-APHIS 2017 Farm Bill
Projects 3.0245.01 and 3.0245.02.

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MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 601-611
https://doi.org/10.5248/134.601

Leucoagaricus brunneus sp. nov.
from Khyber Pakhtunkhwa, Pakistan

ZIA ULLAH"*?, SANA JABEEN?®3, MUHAMMAD FAISAL’,
HaBiB AHMAD‘, ABDUL NASIR KHALID”

' Department of Microbiology and Molecular Genetics & "Department of Botany:
University of the Punjab,
Quaid-e-Azam Campus-54590, Lahore, Punjab, Pakistan

*Department of Botany, Division of Science & Technology,
University of Education, Township, Lahore, Pakistan

“Islamia College, University Peshawar, Peshawar, Pakistan

* CORRESPONDENCE TO: Ziaullah.phd.mmg@pu.edu.pk

ABsTRACT—A field survey of macrofungi from the Hindu Kush range of District Swat
revealed a fungus morphologically similar to Leucoagaricus but representing a new species,
here named L. brunneus. A detailed description and comparison with closely allied taxa are
provided. Maximum likelihood analysis based on the internal transcribed spacer rDNA
region (ITS) confirm its placement in Leucoagaricus sect. Rubrotincti.

Key worps—Agaricaceae, Agaricales, Agaricomycetes, phylogeny, polyphyletic

Introduction

Leucoagaricus Locq. ex Singer, assigned to the family Agaricaceae (Singer
1986, Vellinga 2004), is represented by over 100 species in the northern and
southern hemispheres (Kumar & Manimohan 2009; Ge 2010; Liang & al. 2010;
Vellinga 2010; Vellinga & Balsley 2010; Vellinga & al. 2010; Mufioz & al. 2012,
2014; Kumari & Atri 2013; Malysheva & al. 2013; Ye & al. 2014; Ge & al. 2015;
Qasim &al. 2015; Dovana & al. 2017; Hussain & al. 2018; Usman & Khalid 2018).
From Pakistan only ten Leucoagaricus species have been reported previously:
L. asiaticus Qasim & al., L. badius S. Hussain & al., L. lahorensiformis S. Hussain

602 ... Ullah & al.

& al., L. lahorensis Qasim & al., L. leucothites (Vittad.) Wasser, L. pabbiensis
Usman & Khalid, L. pakistaniensis Jabeen & Khalid, L. serenus (Fr.) Bon &
Boiftard. L. sultanii S. Hussain & al., and L. umbonatus S. Hussain & al. (Ahmad
& al. 1997, Ge & al. 2015, Qasim & al. 2015, Hussain & al. 2018, Usman &
Khalid 2018). The genus is characterized morphologically by small- to medium-
sized basidiomata with entire pileus margins, free lamellae, a central, equal to
bulbous stipe, a membranous, persistent annulus, a white, cream, or pink spore
print white, usually dextrinoid basidiospores, the presence of cheilocystidia,
rare occurrence of pleurocystidia, and absence of clamp connections (Singer
1986).

Several molecular phylogenies indicate that Leucoagaricus is polyphyletic
(Johnson & Vilgalys 1998, Johnson 1999, Vellinga 2004). Vellinga (2003,
2004) showed that Leucoagaricus and Leucocoprinus species cluster together
phylogenetically but intermix within a single clade. Because of the large number
of species in the clade and relatively limited molecular data, the taxonomic
and phylogenetic relationships among many Leucoagaricus and Leucocoprinus
species have not yet been resolved.

During the present investigation, one Leucoagaricus specimen collected
from the Shawar Valley (district Swat, Khyber Pakhtunkhwa, Pakistan) is
proposed here as a new species, L. brunneus, supported by our morphological
and phylogenetic analyses.

Material & methods

The mushroom was collected during a forest foray in Shawar valley, Khyber
Pakhtunkhwa, Pakistan, during the 2017 monsoon season and photographed at the
sampling site. Macrocharacters were recorded from the fresh basidioma, with colors
coded according to Munsell’s Soil Color Charts (1975). The specimen was air dried for
further analyses and deposited in the Herbarium of University of the Punjab, Quaid-
e-Azam Campus, Lahore, Pakistan (LAH).

For microscopic studies, fruiting body tissues were mounted in 2% KOH and
Congo red (to increase contrast). Basidia, basidiospores, cheilocystidia, and pellicular
elements were measured using Scopelmage 9.0. DNA extraction followed Bruns
(1995). The internal transcribed spacer region (ITS1-5.8S-ITS2 = ITS) was amplified
with the ITS1F/ITS4 primer combination (White & al. 1990, Gardes & Bruns 1993).
PCR products were purified and both strands were sequenced by Beijing Genomic
Institute (BGI).

For phylogenetic analysis, an ITS consensus sequence was generated in BioEdit
(Hall 1999) and BLAST searched at NCBI (https://www.ncbi.nlm.nih.gov/);
closely related sequences in L. sect. Rubrotincti were retrieved from both database
and literature (Hussain & al. 2018). Lepiota subgracilis Kihner was chosen as
outgroup (Liang & al. 2010). All sequences were aligned online using PRANK tool

Leucoagaricus brunneus sp. nov. (Pakistan) ... 603

(https://www.ebi.ac.uk/goldman-srv/webprank/). Gaps were treated as missing data.
The final aligned ITS dataset was phylogenetically analyzed through MEGA 6.0
software. Maximum Likelihood (ML) analysis was performed by selecting Kimura
2-parameter with Gamma Distributed model using Best-Fit Substitution Model
approach in MEGA 6.0 (Tamura & al. 2013).

Taxonomy

Leucoagaricus brunneus Z. Ullah, Jabeen & Khalid, sp. nov. FIGS 1, 2
MB 827985
Differs from Leucoagaricus truncatus by its smaller size, smooth shiny non-squamulose

pileus with brown striations, and its longer and narrower elongate to cylindrical
basidiospores.

Type: Pakistan. Khyber Pakhtunkhwa Province, Swat district, Lower Shawar, under
Quercus oblongata D. Don (Fagaceae), 8 July 2017, Zia Ullah LS4 (Holotype, LAH35862;
GenBank MH990662).

EryMotoey: brunneus (Latin) refers to the brown coloration of the pileus fibrils.

Figure. 1. Leucoagaricus brunneus (holotype, LAH35862). Basidioma. Scale bars = 0.5 cm.

604 ... Ullah & al.

Ee

Figure. 2. Leucoagaricus brunneus (holotype, LAH35862). A. Basidiospores; B. Basidia;
C. Cheilocystidia; D. Pileipellis; E. Stipitipellis. Scale bars = 10 um. (Drawing by Sana Jabeen.)

PiLteEus 35 mm diam, plane with incurved margins and umbonate center,
surface smooth and shiny, dark brown (10YR1/2) from center to lighter brown
(10YR2/4) towards margin in the form of radial striations, context cream.

Leucoagaricus brunneus sp. nov. (Pakistan) ... 605

LAMELLAE free, close to crowded, margins entire, cream. LAMELLULAE
absent. STIPE central, 9 x 4-8 mm, narrower towards the apex and wider
(<8 mm) towards the base, then again narrowing (<6 mm) at the base,
cream with grayish brown (10YR4/4) patches over the central part; annulus
superior (at the center of the upper half), white.

BasIDIOSPORES [30/1/1] (8.0—)8.2-10.5(-10.6) x (4-)4.1-4.9(-5.1) um,
Q = (1.8-)1.9-2.1(-2.2), avQ = 2, elongate to cylindrical in face view,
amygdaliform in side view, smooth, germ pore lacking, dextrinoid in
Melzer’s reagent, hyaline in KOH. Basip1a 17.3-19 x 7.4—-8.1 um, clavate,
smooth, hyaline, 4-spored. CHEILOCYSTIDIA 29.7-32.8 x 8.5-12.7 um,
clavate to subclavate, hyaline, without crystals. PLEUROCysTIDIA absent.
PILEIPELLIS a cutis with slightly clavate to cylindrical elements, 4.1-6.7 um
diam, hyaline in KOH. Stiprripexuis cylindric hyphae, 3.0-5.2 um diam,
hyaline in KOH. CLAMP CONNECTIONS absent in all tissues.

EcoLtocy & DISTRIBUTION—Saprobic and solitary on humus-rich
soil under Quercus oblongata [= Q. incana Roxb., nom. illeg.], at 1200 m
elevation, in moist temperate Quercus vegetation.

Phylogenetic analysis

The 72 ITS sequence dataset (TABLE 1, Fic. 3) comprises 757 positions.
Maximum likelihood analysis clusters the local collection in a sister clade with
L. truncatus Z.W. Ge & Zhu L. Yang and L. purpureolilacinus Huijsman with a
bootstrap support of 72% (Fia. 3).

Discussion

Leucoagaricus brunneus can be distinguished from L. truncatus based on
basidioma size. Leucoagaricus truncatus produces medium to large (40-80
mm) basidiomata (Ge & al. 2015) and is further separated by the orange-white
to gray-orange furfuraceous squamules (Ge & al. 2015) on its pileus surface
in contrast to the smooth shiny pileus with brown striations that distinguish
L. brunneus. Leucoagaricus truncatus is further separated microscopically by
its more broadly ovoid and broadly amygdaliform basidiospores (Ge & al.
2015). The ITS sequence analysis also provides strong bootstrap support for
L. brunneus as an independent taxon.

Leucoagaricus brunneus differs from L. purpureolilacinus, which is
characterized by a pinkish brown pileus and presence of crystalliferous
cheilocystidia (Vellinga 2001).

Pileus morphology also separates Leucoagaricus brunneus with its dark
brown umbo and brownish striations running towards the margin from

606 ... Ullah & al.

the center from L. serenus and L. crystallifer Vellinga, both characterized
by whitish basidiomata and pilei with white-to cream obtuse umbos with
obvious striations near the margins. Leucoagaricus serenus and L. crystallifer
are further distinguished by their ovoid and broadly amygdaliform

TABLE 1. Leucoagaricus species and specimens, and Lepiota subgracilis outgroup, used
for ITS phylogenetic analyses.

SPECIES VOUCHER COUNTRY GENBANK NO.
L. americanus Vellinga 2454 (UCB) USA AY 176407
JRH091509-1 (TENN) USA MF773593
L. asiaticus LAH5872011 Pakistan KP 164972
LAH10012012 Pakistan KP 164971
L. badius LAH SH210 Pakistan KU647734
LAH SH148 Pakistan KU647736
L. bresadolae Bas7981 USA AF295929
MCVE:756 Italy GQ329047
CCBAS802 Czech Rep. LN714565
L. brunneus LAH35862 [T] Pakistan MH990662
L. crystallifer Huijser (L) Germany AF482863
SFC 1010003-02 Spain KY350216
L. dyscritus Vellinga 3532B (UC) USA GU136181
Vellinga 3956 (UC) USA GU136180
L. gaillardii MCVE:16517 Italy GQ329064
MCVE:736 Italy GQ329042
L. griseodiscus MCVE:13719 Italy GQ329059
L. japonicus J. Li221 China KY039572
L. jubilaei Guinberteau 99101101 France AY243635
10115A USA KX258658
L. lacrymans Zhang 599 China KY039574
L. lahorensiformiS FH-SHL2 Pakistan KU647730
LAH SHL2 Pakistan KU647729
L. littoralis MCVE:856 Italy GQ329051
MCVE:702 Italy GQ329041
MCVE:13721 Italy GQ329060
L. cf. majusculus MFLU 09-0164 Thailand HM488764
L. medioflavoides MCVE:2324 Italy GQ329055
L. meleagris IMG 1671 USA KY680786
Vellinga 2095 (L) Netherlands AF482867
Vellinga 1990 (L) Netherlands AY176419
CAW-9 India GQ249888

L. menieri
L. nivalis

L. pakistaniensis

L. purpureolilacinus

L. rubroconfusus

L. rubrotinctus

L. sardous

Leucoagaricus sp.

L. subcrystallifer
L. sublittoralis

L. subpurpureolilacinus

L. subvolvatus

L. sultanii

L. truncatus

L. umbonatus

L. vassiljevae

L. viscidulus

L. volvatus

Lepiota subgracilis

herb. Huijser

Yang 5792

LAH SJF13

LAH SJF23
MCVE:754
Vellinga 2291 (L)
ZT13003 (ZT)
gr0557

gr157

KUN:HKAS 54317
KUN:HKAS 54240
AC5195

Thiele 2646
JZB2115002

BAB 4737

Ge 97

Yang 3972

Vellinga 2561
TENN:070790
FLAS-F-60259
M.M. Rogers (UCB)
Vellinga 2484 (UC)
Ge 878

Ecv2235

Yang 3959

Ge 406
Brand s.n. (L)
LAH SH115b
LAH SH115
Ge 793

LAH SHL1
LAH SHL8
LE 10350

LE 289338
LE 289432
AC4187
AC1785
Vellinga 1783 (L)
HKAS 5802

Netherlands
China
Pakistan
Pakistan
Italy
Netherlands
USA

China
China
China
China
China

Spain
Australia
China

India

China
China

USA

USA

USA

USA

USA

China
Netherlands
China

China
USA
Pakistan
Pakistan
China
Pakistan
Pakistan
Russia
Russia
Russia
Spain
Spain
Netherlands
China

Leucoagaricus brunneus sp. nov. (Pakistan) ... 607

KP300879
KY039573
KU647727
KU647728
GQ329045
AF482869
KP300875
KP300877
KP300876
JN944082

JN944081

JX827166

KT992149
AY176432
JN907015

KR154966
KP096237
KP096238
AY176430
MF686514
MF153051
AY176434
GU136182
KP096236
AY176442

KP096234

KP096233
KP300878
KU647732
KU647733
KP096235
KU647737
KU647738
JX133169

JX133170

JX896447

KT992148
KT992150
AY176490
EU416290

608 ... Ullah & al.

69 KP300877 L. rubrotinctus
AY 176442 L. sublittoralis
KP300876 L. rubrotinctus
69 | JX133169 L. vassiljevae
KY 039572 L. japonicus
JX133170 L. vassilievae
JX896447 L. vassilievae
96 |75 JN944082 L. rubrotinctus
GQ329045 L. purpureolilacinus
99 KU647730 L. lahorensiformis
KU647729 L. lahorensiformis
4 JN944081 L. rubrotinctus
V1 KP300875 L. rubroconfusus
50 JX827166 L. rubrotinctus
63 AY 176432 Leucoagaricus sp.
99 | KP096234 L. subpurpureolilacinus
KP096233 L. subpurpureolilacinus
93 | KU647727 L. pakistaniensis
KU647728 L. pakistaniensis
92| P24 JN907015 Leucoagaricus sp.
99 KR154966 Leucoagaricus sp.
98 /KU647734 L. badius
KU647736 L. badius
99 — KP164972 L. asiaticus

KP164971 L. asiaticus
KT992148 L. viscidulus
90 99 KU647732 L. sultanii

66 50 KU647733 L. sultanii
I 99 — AF482863 L. crystallifer
3p KY350216 L. crystallifer
hg -— KP300879 L. menieri
g, KP300878 L. subvolvatus
4 KT992150 L. volvatus
46 KP096236 L. subcrystallifer
KT992149 L. sardous
99 | GQ329051 L. littoralis
GQ329041 L. littoralis

27 GQ329060 L. littoralis
95 | KP096237 Leucoagaricus e
45 Q329059 L. griseodiscus
KP096238 Leucoagaricus sp.
KY 039573 L. nivalis
78 99 KU647737 L. umbonatus
53 KU647738 L. umbonatus
52 AY 176430 Leucoagaricus sp.
99 |MF686514 Leucoagaricus sp.
MF 153051 Leucoagaricus sp.
51 AF482869 L. purpureolilacinus
Se ? KP096235 L. truncatus
@ MH990662 L. brunneus
AY 176434 Leucoagaricus sp.
99 AY243635 L. jubilaei
18 KX258658 L. jubilaei
is [197 ,GU136181 L. yee
53 GU136180 L. dyscritus
GU 136182 Leucoagaricus sp.
99 ,GQ329064 L. gaillardii
56 GQ329042 L. gaillardii
GQ329055 L. medioflavoides
“4 KY039574 L. lacrymans

4 og | KY680786 L. meleagris

AF 482867 L. meleagris

50 AY 176419 L. meleagris

GQ249888 L. meleagris
84 HM488764 L. cf. majusculus
AY 176407 L. americanus
81 | _ AF295929 L. bresadolae
56} GQ329047 L. bresadolae
MF773593 L. americanus
LN714565 L. bresadolae

99 ,AY176490 Lepiota subgracilis
—| £U416290 Lepiota subgracilis

-——XH
0.02

Ficure. 3. Molecular phylogenetic analysis of ITS sequences of Leucoagaricus species, with Lepiota
subgracilis outgroup. The evolutionary history was inferred by using the Maximum Likelihood
method based on the Tamura 3-parameter model. The analysis involved 72 nucleotide sequences.
All positions containing gaps and missing data were eliminated. There were a total of 448 positions
in the final dataset. Our new species is marked with @.

Leucoagaricus brunneus sp. nov. (Pakistan) ... 609

basidiospores, contrasting with the elongated to cylindric and amygdaliform
basidiospores of L. brunneus. In addition, in L. crystallifer the cheilocystidia
have obvious crystals on the surface (Vellinga 2000, 2001) unlike the smooth
cheilocystidia of L. brunneus.

In comparison with Pakistani Leucoagaricus species, L. brunneus differs
from L. asiaticus characterized by oblong to ellipsoid spores and cheilocystidia
with crystals at the apex (Ge & al. 2015). Morphologically, its pileus color
distinguishes L. brunneus (brown) from L. badius (red), L. pakistaniensis
(whitish), L. lahorensis (dark reddish brown), L. lahorensiformis (light
orange), L. umbonatus (yellowish pink), and L. sultanii (dark yellow) (Qasim
& al. 2015, Hussain & al. 2018). Basidiospore morphology also separates
these species from L. brunneus (Qasim & al. 2015, Hussain & al. 2018). All
the earlier named Pakistani species have smaller spores except L. lahorensis,
whose spores measure more or less the same as L. brunneus (Qasim &
al. 2015), and none except L. brunneus present the same basidiospore
shape—elongate to cylindrical in face view and amygdaliform in profile:
amygdaliform to oblong in L. badius (6.5-7.5 x 4-5 um), ellipsoid to rarely
amygdaliform in L. pakistaniensis (7.5-8 x 4.5-5 um), broadly ellipsoid to
ellipsoid in L. lahorensis (8-10.6 x 6.4—7.6 um), fusiform to amygdaliform
in L. lahorensiformis (6.5-7.5 x 3.5-4 um), amygdaliform to ellipsoid
in L. umbonatus (5.5-6.5 x 3.5-4 um), and amygdaliform to ellipsoid in
L, sultanii (5.5-7 x 3.5-4.5 um) (Qasim & al. 2015, Hussain & al. 2018). The
crystals on the cheilocystidial apices in L. lahorensiformis and L. sultanii also
separate both species from L. brunneus.

Therefore a combination of morphological differences and a phylogenetic
bootstrap support of 72% (Fic. 3) provide convincing support for our new
species, Leucoagaricus brunneus.

Acknowledgments

The work is funded by HEC Research Project number 20-3383/HEC/R&D/14/184.
Sincere thanks to Dr. Chang-Lin Zhao (Forestry College, Southwest Forestry
University, Yunnan, P.R. China) and Dr. Shah Hussain (Center for Plant Sciences and
Biodiversity, University of Swat, Pakistan) for presubmission review of the manuscript.
Their comments and suggestions greatly helped to improve the document. We are
thankful to Sheraz Khan, Numan Fazal, and Niaz Ali for their help in sampling.

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Liang JF, Yang ZL, Xu J, Ge ZW. 2010. Two new unusual Leucoagaricus species (Agaricaceae)
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MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 613-618
https://doi.org/10.5248/134.613

Dictyostelids from Jilin Province, China 3:
new Cavenderia and Dictyostelium records

Pu Liu', SHUNHANG ZHANG', YUE ZOU’, XUEPING KANG’, Yu LI"

' Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi,
Jilin Agricultural University, Changchun, 130118, P. R. China
?Yanbian Academy of Agricultural Sciences, Yanji 133001, P. R. China

* CORRESPONDENCE TO: fungi966@126.com

ABSTRACT—Cavenderia parvispora, Dictyostelium vermiforme, and D. dimigraforme, isolated
from samples of soil collected in Jilin Province, China, represent new records from China.
Descriptions and illustrations based on these isolates are provided.

Key worps—Cavenderiaceae, cellular slime molds, Dictyosteliaceae, Mycetozoa, taxonomy

Introduction

Dictyostelid cellular slime molds (dictyostelids), with both animal-like
(protozoan) and fungus-like characteristics, are primarily inhabitants of the
soil and leaf litter/humus zone of fields and forests, along with animal dung,
where they feed mostly on bacteria. These organisms play an essential role in
maintaining the balance that exists between bacteria and other soil organisms
(Singh 1947; Cavender & Raper 1965a,b; Raper 1973, 1984). Traditional
morphological classifications distributed dictyostelids among four genera
based on differences in sorophore structure and branching patterns (Kirk &
al. 2008). However, a new classification based on unique 18S rRNA sequence
signatures was proposed by Sheikh & al. (2018).

Jilin Province is situated in the temperate zone of China and is characterized
by a monsoon climate (Liu & Li 2014). Previously, a total of 15 species, including
two new dictyostelid species, have been reported from Jilin Province (He & Li

614 ... Liu & al.

2008; Liu & Li 2014, 2017). We report another three species in Cavenderia and
Dictyostelium for the first time from China.

Materials & methods

SAMPLING, ISOLATION, CULTIVATION—Samples of soil were collected from three
localities in Jilin Province in 2008 and 2009. Each 10-20 g soil sample was placed in a
sterile whirl-pack plastic bag for isolation according to Cavender & Raper (1965a). Each
sample was weighed and enough ddH,O added for an initial dilution of 1:10. A 0.5 mL
aliquot of this dilution was added to each of five duplicate culture plates prepared with
hay infusion agar (Raper 1984). After c. 0.4 mL of a heavy suspension of the bacterium
Escherichia coli was added to each culture plate, the plates were incubated at 23 °C with
a 12h light : 12 h dark cycle. Each plate was examined at least once daily for two weeks
after the appearance of initial aggregations. Each isolate recovered from one of the plates
was purified and cultivated for taxonomic studies. Spores from these isolates were frozen
in HL 5 media (Cocucci & Sussman 1970) and stored at —80 °C in the herbarium of the
Mycological Institute of Jilin Agricultural University, Changchun, China (HMJAU).

OBSERVATION—The isolates were identified using morphological descriptions
(Raper 1984) and molecular characteristics (Sheikh & al. 2018). After we marked the
location of each early aggregating clone and sorocarp in a plate, we observed life cycle
stages under a Zeiss Axio Zoom V16 dissecting microscope with a 1.5x objective
and a 10x ocular. Sorocarps were mounted in water on slides for observation and
measurements of spores, sorophores, and sorocarps using a Zeiss Axio Imager A2 light
microscope mounted with 10x ocular and 10, 40, and 100x (oil) objectives. Photographs
were taken with Zeiss Axiocam 506 color microscope camera.

Taxonomy

Cavenderia parvispora (H. Hagiw.) S. Baldauf, S. Sheikh & Thulin,
Protist 169(1): 20. 2018. PLATE 1A-D

When cultured at 23 °C on non-nutrient agar with E. coli, sorocarps
gregarious or solitary, unbranched or sparsely irregularly branched, sometimes
prostrate, phototropic, normally 0.2-4.4 mm long. Sorophore colorless, sinuose,
slender, tapering from bases to tips, consisting of one tier of cells except for
the in base and tip, bases clavate, tips clavate. Sori white, globose, commonly
30-150 um diam. Spores hyaline, elliptical, 3.7-6.0 x 1.9-2.9 um, with a few
conspicuous polar granules. Aggregations with radiate streams or mound-like.

SPECIMEN EXAMINED: CHINA, JILIN PROVINCE, Zuojjia, isolated from soil ($0425) from

a broadleaf forest, 9 Sep. 2008 (HMJAU MR056).
COMMENTS: Cavenderia parvispora was first isolated from forest soil sampled in
Japan (Hagiwara 1989). Its medium-sized sorocarps, thin delicate sorophores
with clavate tips, and small elliptical spores readily distinguish this species
from morphologically similar dictyostelids.

Cavenderia and Dictyostelium spp. new from Jilin (China) ... 615

4

198 @ OVE t.
PEGI @ o® =

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a” ee cy “Fry “aK A/ QO see ee
if : _ pee “| ee! Cy =e : .

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PLATE 1. Cavenderia parvispora (HMJAU MR056): A. Sorocarps; B. Sorophore base;
C. Sorophore tip; D. Spores. Dictyostelium vermiforme (HMJAU MR058): E. Sorocarps;
F, G. Vermiform and curved sorogens; H. Sorophore base; I. Sorophore tip; J. Spores. Scale bars:
A, E= 2 mm; B, C, H, 1 =5 um; D,J = 10 um; KF G=1 um.

616 ... Liu & al.

Dictyostelium vermiforme Vadell & Cavender, Mycologia 99: 118. 2007. PLATE 1E-J
When cultured at 23 °C on non-nutrient agar with Escherichia coli, sorocarps

clustered or solitary, normally prostrate, 1.0-2.0 mm long. Sorophore colorless,
tapering from bases to tips, consisting of one tier of cells except for the base
and tip, bases small round or expanded, tips capitate or expanded. Sori white,
globose, commonly 50-130 um diam. Spores hyaline, oblong to elliptical,
4.7-7.0 x 2.9-3.9 um, with sparse or prominent polar granules. Aggregations
with flattened streams, forming mound-like early sorogens. Sorogens
developing rapidly, becoming vermiform and curved, migrating freely to form
sinuous and curled late sorogens with prostrate sorophores.

SPECIMEN EXAMINED: CHINA, JILIN PROVINCE, National Nature Reserve, isolated

from soil (S0083-1) from a broadleaf forest, 10 Jun. 2009 (HMJAU MR058).
ComMENTs: Dictyostelium vermiforme was originally isolated from soil/litter
sampled in Iguazu Regional Park, Argentina (Vadell & Cavender 2007). This
species is characterized by its vermiform and curved early migrating sorogens.

Dictyostelium dimigraforme Cavender, J. Gen. Microbiol. 62:115.1970. | PLATE 2
When cultured at 23 °C on non-nutrient agar with Escherichia coli, sorocarps

normally solitary, unbranched, erect or inclined, 3.5-10.5 mm long. Sorophore
colorless or slightly yellow, tapering from bases to tips, consisting of one tier of
cells except for the base and tip, bases round or slightly enlarged, tips capitate.
Sori globose, off-white to yellow, 220-380 um diam. Spores hyaline, oblong
to elliptical, 7.7-12.0 x 2.9-4.9 um, without polar granules. Aggregations
radiating.

SPECIMEN EXAMINED: CHINA, JILIN PROVINCE, Chanchung, Jingyuetan National

Natural Park, isolated from soil (S0446) from a broadleaf forest, 10 Sep. 2008 (HMJAU

MRO059).
ComMENTs: Dictyostelium dimigraforme was isolated the first time from surface
soil sampled in a tropical forest in the West Indies (Raper 1984). This species is
characterized by its yellow sori and round bases, and can be distinguished from
D. discoideum by its lack of basal disk.

Discussion

Our study has raised to 18 the number of dictyostelid species known from
Jilin Province. Jilin Province is located in the temperate zone which has rich
forests optimal for dictyostelids. The three dictyostelid species (Cavenderia
parvispora, Dictyostelium vermiforme, and D. dimigraforme) reported here do
not appear to be distributed worldwide (Hagiwara 1989, Vadell & Cavender
2007, Raper 1984). In particular, Dictyostelium vermiforme (originally isolated

Cavenderia and Dictyostelium spp. new from Jilin (China) ... 617

ra €

PLATE 2. Dictyostelium dimigraforme (HMJAU MR059): A, B. Sorocarps; C. Sorophore base;
D. Sorophore tip; E. Spores. Scale bars: A, B = 2 mm; C, D =5 um; E= 10 um.

618 ... Liu & al.

from c. 26°S in South America) and D. dimigraforme (originally isolated from
c. 11°N in the West Indies) have now been isolated from c. 44°N in Asia. Clearly,
the dictyostelids of Jilin Province need to be investigated in more detail.

Acknowledgments

We wish to express our appreciation to peer reviewers Prof. Steven L. Stephenson
(University of Arkansas, U.S.A.) and Prof. John C. Landolt (Shepherd University, U.S.A.)
for their valuable comments relating to this manuscript. This study was supported
by the National Natural Science Foundation of China (No. 31870015, 31300016),
the Science and Technology Research Programs of the Education Department of
Jilin Province in the Thirteenth Five-Year Plan (No. JJKH20180671KJ), Science and
Technology Development Program of Jilin Province (No. 20180101273JC), and 111
Project (No. D17014).

Literature cited

Cavender JC, Raper KB. 1965a. The Acrasieae in nature. I. Isolation. American Journal of
Botany 52: 294-296. https://doi.org/10.2307/2439943

Cavender JC, Raper KB. 1965b. The Acrasieae in nature. II. Forest soil as a primary habitat.
American Journal of Botany 52: 297-302. https://doi.org/10.2307/2439944

Cocucci SM, Sussman M. 1970. RNA in cytoplasmic and nuclear fractions of cellular slime
mold amoebas. Journal of Cell Biology 45: 399-407. https://doi.org/10.1083/jcb.45.2.399

Hagiwara H. 1989. The taxonomic study of Japanese dictyostelid cellular slime molds. National
Science Museum, Tokyo.

He XL, Li Y. 2008. A new species of Dictyostelium. Mycotaxon 106: 379-383.

Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Dictionary of the fungi, 10th edition.
CABI, UK.

Liu P, Li Y. 2014. Dictyostelids from Jilin Province, China. I. Phytotaxa 183(4): 279-283.
https://doi.org/10.11646/phytotaxa.183.4.7

Liu P, Li Y. 2017. Dictyostelids from Jilin Province, China. II. Phytotaxa 323(1): 77-82.
https://doi.org/10.11646/phytotaxa.323.1.6

Raper KB. 1973. Acrasiomycetes. 9-36, in: GC Ainsworth &al. (eds). The Fungi, vol. IVB.
Academic Press, Inc. New York.

Raper KB. 1984. The dictyostelids. Princeton University Press, Princeton.

Sheikh S, Thulin M, Cavender JC, Escalante R, Kawakami S, Lado C, Landolt JC, Nanjundiah
V, Queller DC, Strassmann JE, Spiegel FW, Stephenson SL, Vadell EW, Baldauf SL. 2018. A
new classification of the dictyostelids. Protist 169(1): 1-28.
https://doi.org/10.1016/j.protist.2017.11.001.

Singh BN. 1947. Studies on soil Acrasieae. 1. Distribution of species of Dictyostelium in soils
of Great Britain and the effects of bacteria on their development. Journal of General
Microbiology 28: 417-429. https://doi.org/10.1099/00221287-1-1-11

Vadell EM, Cavender JC. 2007. Dictyostelids living in the soils of the Atlantic Forest, Iguazu
Region, Misiones, Argentina: description of new species. Mycologia 99(1): 112-124.
https://doi.org/10.1080/15572536.2007.11832606

MYCOTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 619-625
https://doi.org/10.5248/134.619

Records of Aureobasidium harposporum, Sarcophoma miribelii,
and Stigmina dothideoides from Turkey

MAKBULE ERDOGDU’, MERVE ULUKAPT?,
ALI IHSAN KARAYEL?, ZEKIYE SULUDERE?

' Department of Landscape Architects, Faculty of Agriculture &
? Graduate School of Natural and Applied Sciences:
Ahi Evran University, Bagbas1, Kirsehir, Turkey
* Department of Biology, Faculty of Science, Gazi University,
Teknikokullar, Ankara, Turkey

* CORRESPONDENCE TO: merdogdu@ahievran.edu.tr

ABsTRACT—During field studies on the microfungi in Mucur District of Kirsehir Province,
Aureobasidium harposporum, and Stigmina dothideoides were revealed as new records for
Turkey; and Sarcophoma miribelii, previously recorded from northeastern Turkey, is reported
from central Turkey. Distinguishing morphological characters of these species are described,
and their photographs are provided.

Key worps—Ascomycota, Dothideales, leaf pathogenic fungi, Mycosphaerellales, SEM

Introduction

Aureobasidium Viala & G. Boyer (Saccotheciaceae, Dothideales)
contains 24 species (Index Fungorum 2018). The genus is characterized by
synchronous conidium production on hyaline conidiogenous cells. Species of
Aureobasidium produce variously shaped one-celled conidia from terminal,
lateral, or intercalary hyaline conidiogenous cells. Members of this genus
occur as saprophytes on a variety of substrates or as parasites on phanerogams
(Hermanides-Nijhof 1977).

Sarcophoma Hohn. (Dothioraceae, Dothideales) was proposed by Hohnel
in 1906 as a monotypic genus, but four species are now accepted (Index
Fungorum 2018).

620 ... Erdogdu & al.

Stigmina Sacc. (Mycosphaerellaceae, Mycosphaerellales) was established
by Saccardo in 1880; c. 80 species are currently accepted in the genus (Index
Fungorum 2018). In Turkey, the Stigmina species are poorly known and have
not yet been intensively studied; of the four species reported from Turkey—
S. carpophila, S. compacta, S. obtecta, S. platani—(Gdobelez 1964, Hiiseyin &
al. 2003, Cimen & Ertugrul 2007, Erdogdu & Huseyin 2008), two have been
transferred to other genera: S. compacta [= Thyrostroma compactum] and
S. platani [= Pseudocercospora platanigena].

Materials & methods

Host specimens were prepared following conventional herbarium techniques.
Host plants were identified using the Flora of Turkey and East Aegean Islands (Davis
1965-85). Thin fungal sections prepared from host tissue were examined under a
Leica DM E light microscope and measured from mounts in tap water. Infected
host surfaces were photographed using a Leica EZ4D stereomicroscope. The fungi
were identified using relevant literature (for Aureobasidium: Saccardo 1892 (as
Gloeosporium), Hermanides-Nijhof 1977; for Sarcophoma: Saccardo 1884 (under
Phoma), Morgan-Jones 1971, Aa 1975; for Stigmina: Ellis 1959, Shoemaker & Egger
1982). All examined specimens were deposited in the Mycology Laboratory of Ahi
Evran University, Department of Biology, Kirsehir, Turkey (AEUT).

For scanning electron microscopy (SEM), 8-10 mm square pieces of infected
leaf were mounted on aluminium stubs with double-sided adhesive tape. They were
coated with gold using Polaron SC 502 Sputter Coater and were examined with
Jeol JSM 6060 scanning electron microscope operated at 5-10 kV in the Electron
Microscopy Unit, Faculty of Science, Gazi University, Ankara, Turkey.

Taxonomy

Aureobasidium harposporum (Bres. & Sacc.) Herm.-Nijh.,
Stud. Mycol. 15: 151 (1977) PLATE 1

Spots visible on both sides of leaves and twigs, generally leaves beginning
to dry from the tip, brown on the upper surface, fuscous chestnut on the lower
surface, spots margin dark brown; infected leaves dropping prematurely from
the shrub. Conrp1omarta acervular, subepidermal, yellowish, broadly elliptical,
196-330 um diam. CONIDIOGENOUS CELLS erect, densely packed, clavate to
subcylindrical, 10-20 x 3.5-6.5 um, forming conidia simultaneously at the
apex. Conip1 one-celled, falcate, sometimes elliptical or slightly curved, oil
droplet, smooth-walled, 13.7-19.3(-21.8) x 4.4-5.4 um, hyaline.

SPECIMEN EXAMINED—TURKEY, Kirsenrr, Mucur District, Tekken Village,
39°02'13”N, 34°13’08”E, 977 m asl, on living leaves and stems of Viscum album L. subsp.
album (Santalaceae), 19.06.2013, M. Ulukap1 (AEUT MU1050).

Aureobasidium, Sarcophoma, Stigmina spp. in Turkey ... 621

we

10k \ xS"6Ge— ) Sam GUFEF | | {OkU ~~ x4,388 Sum GuFEF

PLATE 1. Aureobasidium harposporum (AEUT MU1050). A. leaf spots; B. acervuli on leaf;
C. acervuli on leaf (SEM); D, E. acervulus on leaf (SEM); E acervulus, vertical section; G. conidia;
H, I. conidia (SEM).

Notes: Viscum album (European mistletoe) is an evergreen, perennial,
epiphytic, hemiparasitic shrub that lives on a wide range of woody plant species
(Zuber 2004). European mistletoes can affect their host trees in many ways.
Known effects from mistletoe infection include lowering the vigor of the host,
inducing premature mortality, reducing quality and quantity of wood grown,
reducing fruit production, and predisposing trees to secondary infection by
other agents, such as insects or decay fungi (Hawksworth 1983).

Biological control of parasites by using plant pathogens has gained
acceptance as a practical, safe, and environmentally beneficial management
method applicable to agro-ecosystems (Charudattan 2001). The use of biological
control agents for weed control has attracted increased attention (Ozaslan &
al. 2013; Ozaslan 2016), and diagnosing host weeds and their natural enemies
is the foundation stone of a successful biological control program (Ozaslan
2016). Control of European mistletoe is an important consideration for the
forest service in Turkey (Yiiksel & al. 2005). Over 20 microscopic fungi live on
European mistletoe, but only a few of them cause major damage to the plant

622 ... Erdogdu & al.

(Karadzic & al. 2004). Of these, Aureobasidium harposporum, which causes leaf
spot disease of European mistletoe, appears to have potential as a biological
control agent against of this semi-parasite. In this study, Aureobasidium
harposporum on living leaves and stems of Viscum album subsp. album is
reported as new to the mycobiota of Turkey.

The Turkish specimen agrees with other reports of Aureobasidium
harposporum in conidiomata and conidia morphology, the only observable
difference being the smaller dimensions of acervuli and conidia. Saccardo
(1892) describes 200 um diam. acervuli and 18-20 x 4-5 um conidia, while
Hermanides-Nijhof (1977) cites 250-500 um acervuli and (16-)17-21(-23) x
3.5-5 um conidia.

XZ66 160)tmh

X7> 566 Zharn

Aureobasidium, Sarcophoma, Stigmina spp. in Turkey ... 623

Sarcophoma miribelii (Fr.) Héhn., Hedwigia 60: 133 (1918) PLATE 2
= Macrophoma miribelii (Fr.) Berl. & Voglino, Atti Soc.
Veneto-Trent. Sci. Nat.10(1): 179 (1886)

PYcNIDIA numerous, hypophyllous (rarely epiphyllous), at first covered
by the epidermis, later becoming erumpent, scattered, 143-289 um diam.,
yellowish or brown. Conrp1a single-celled, ovoid, rounded at apex, attenuate
at the base, smooth-walled, (8.4—)10-13.6(-14.3) x 6.2-8.7 um, hyaline,
with granular contents.

SPECIMEN EXAMINED—TURKEY, Kirseuir, Mucur District, Seyfegolti, 39°06’58’N,

34°12'11”E, 1135 m asl, on living leaves and stems of Buxus sempervirens L. (Buxaceae),

16.04.2014, M. Ulukapi (AEUT MU1099).
Notes: Our Turkish specimen is morphologically similar to Sarcophoma
miribelii specimens described in the literature (Saccardo 1884, Morgan-Jones
1971, Aa 1975). Sarcophoma miribelii has previously been reported (without
description or illustration) from Rize Province in coastal northeastern
Turkey (Gobelez 1964, Hiiseyin & al. 2005; both as Macrophoma mirbelii
[sic]), whereas our new record is from Kirsehir Province in central Turkey at
c. 1100 m asl. Sarcophoma miribelii is found worldwide, wherever the Buxus
host occurs (Aa 1975).

Stigmina dothideoides (Ellis & Everh.) M.B. Ellis, Mycol. Pap. 72: 53 (1959) PLATE 3

SPORODOCHIA erumpent through epidermis, pustulate, 1-4 mm
diam., circular to elliptical, blackish. CoNIDIOPHORES densely crowded,
arising from stromatic hyphae, pale brown to medium brown, cylindrical
to lageniform, 1-2- septate, often branched at the base. Conip1a blastic,
solitary and terminal, elliptical to obovoid, at first one-celled, becoming
3-septate, non-constricted or slightly constricted at the septum, rounded
at apex, truncated to obtusely rounded at the base, smooth-walled, 25-38 x
11.8-13.9(-14.5) um, golden brown.

SPECIMEN EXAMINED— TURKEY, Kirsenir, Mucur District, Gumtiskiimbet Village,

39°05'52”N, 34°12’26”E, 1180 m asl, on stems of Artemisia sp. (Asteraceae), 16.04.2014,

M. Ulukapi (AEUT MU1087).
Notes: The species was first described by Ellis & Everhart (1891, as
Clasterosporium dothideoides), who observed symptoms on stems of
Artemisia cana and Shepherdia argentea in Montana, USA; and it has

PLATE 2 (left). Sarcophoma miribelii (AEUT MU1099). A. pycnidia on leaf; B. pycnidia on
leaf (SEM); C. pycnidium on leaf (SEM); D. pycnidium, vertical section; E. conidia; F. conidia
(SEM).

624 ... Erdogdu & al.

15 um

Cp - ls

PLATE 3. Stigmina dothideoides (AEUT MU1087). Top Lert. conidia and conidiophores; Top
RIGHT. conidium and conidiophore; BoTTom. conidia.

been recorded on Gaillardia aristata further north in Alberta, Canada
(Shoemaker & Egger 1982). Stigmina dothideoides on branches of
Artemisia sp. is reported as new to the mycobiota of Turkey.

Although our Turkish specimen of Stigmina dothideoides is
morphologically similar to specimens described in the literature,
it differs slightly in having shorter and narrower 3-septate conidia,
compared with 25-46 x 13-19 um 3-septate conidia in Ellis (1959) and
30-38(-42) x (12.3-)14-16.8 um 3(—4)-septate conidia in Shoemaker
& Egger (1982).

Acknowledgments

The authors thank Sevda Kirbag (Department of Biology, Firat University, Elazig,
Turkey) and Cumali Ozaslan (Department of Plant Protection, Dicle University,
Diyarbakir, Turkey) for pre-submission review. This work was supported by the
Ahi Evran University Scientific Research Projects Coordination Unit. (Project
Number: PYO-Fen.4003.13.005 and FEF.A4.17.006).

Aureobasidium, Sarcophoma, Stigmina spp. in Turkey ... 625

Literature cited

Aa HA van der. 1975. The perfect state of Sarcophoma miribelii. Persoonia 8(3): 283-289.

Charudattan R. 2001. Biological control of weeds by means of plant pathogens: significance
for integrated weed management in modern agro-ecology. Biocontrol 46(2): 229-260.
https://doi.org/10.1023/A:101147753

Cimen I, Ertugrul BB. 2007. Determination of mycoflora in almond plantations under drought
conditions in Southeastern Anatolia project Region, Turkey. Plant Pathology Journal 6(1):
82-86. https://doi.org/10.3923/ppj.2007.82.86

Davis PH (ed.). 1965-85. Flora of Turkey and East Aegean Islands. Vols 1-9. Edinburgh
University Press, Edinburgh.

Ellis MB. 1959. Clasterosporium and some allied dematiaceae—phragmosporae. II. Mycological
Papers 72.75 p.

Ellis JB, Everhart BM. 1891. New species of fungi from various localities. Proceedings of the
Academy of Natural Sciences of Philadelphia 43: 76-93.

Erdogdu M, Huseyin E. 2008. Microfungi of Kurtbogazi1 Dam (Ankara) and its environment. Ot
Sistematik Botanik Dergisi 14(1): 131-150.

Gébelez M. 1964. La mycoflore de Turguie. (List of fungi of Turkey). II. Mycopathologia et
Mycologia Applicata 23(1): 47-67. https://doi.org/10.1007/BF02049185

Hawksworth FG. 1983. Mistletoes as forest parasites. 317-333, in: M Calder, P Bernhardt (eds).
The biology of mistletoes. Australia, Academic Press.

Hermanides-Nijhof EJ. 1977. Aureobasidium and allied genera. Studies in Mycology. 15: 141-177.

Huseyin E, Selcuk FE, Gaffaroglu M. 2003. Some materials on mitosporic fungi from Turkey I.
Hyphomycetes. Botanica Lithuanica 9(2): 151-160.

Hiiseyin E, Selcuk F, Gaffaroglu M. 2005. Materials on the micromycetes on box tree (Buxus) and
Rhododendron from Turkey. 62-68, in: Proceedings of the XVI Symposium of Mycologists
and Lichenologists of Baltic States. 21-25 September, 62-68, Cesis, Latvia.

Index Fungorum 2018: http://www.indexfungorum.org. [accessed September 2018].

Karadzi¢ D, Lazarev V, Milenkovi¢ M. 2004. The most significant parasitic and saprophytic
fungi on common mistletoe (Viscum album L.) and their potential application in biocontrol.
Bulletin Faculty of Forestry, University of Bajna Luka, Serbia 89: 115-126.

Morgan-Jones G. 1971. Conidium ontogeny in coelomycetes. I. Some amerosporous
species which possess annellides. Canadian Journal of Botany 49(11): 1921-1929.
https://doi.org/10.1139/b71-267

Ozaslan C, Hiiseyin E, Erdogdu M. 2013. Microfungi species on the weeds of agro-ecosystem
(wheat ecosystem) in Adtyaman City. Mantar Dergisi 4(2): 10-18.

Ozaslan C. 2016. Downy mildews species on the weeds of lentil fields in Diyarbakir in Turkey.
Scientific Papers. Series A. Agronomy 59: 365-367.

Saccardo PA. 1884. Sylloge Spheropsidearum et Melanconiarum. Sylloge Fungorum 3. 860 p.

Saccardo PA. 1892. Supplementum universale, pars II. Discomyceteae-Hyphomyceteae. Sylloge
Fungorum 10. 964 p.

Shoemaker RA, Egger KN. 1982. Stigmina dothideoides. Fungi Canadenses 212. 2 p.

Yuksel B, Akbulut S, Keten A. 2005. The damage, biology and control of pine mistletoes (Viscum
album ssp. austriacum (Wiesb.) Vollman). Turkish Journal of Forestry 2: 111-124.

Zuber D. 2004. Biological flora of Central Europe: Viscum album L. Flora 199(3): 181-203.
https://doi.org/10.1078/0367-2530-00147

MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 627-632
https://doi.org/10.5248/134.627

Bactrodesmium pulcherrimum sp. nov.
from Ecuador

FERNANDO ESPINOZA’, DAYNET SOSA’, LIZETTE SERRANO’,
ADELA QUEVEDO’, FREDDY MAGDAMA’, MARCOS VERA’,
SIMON PEREZ-MARTINEZ’, ELAINE MALOSSO},

RAFAEL F, CASTANEDA-Rut1Iz?*

' Escuela Superior Politécnica del Litoral, ESPOL, (CIBE),
Campus Gustavo Galindo Km. 30.5 Via Perimetral,
PO. Box 09-01-5863, Guayaquil, Ecuador
? Universidad Estatal de Milagro (UNEMI), Facultad de Ingenieria,
Cdla. Universitaria Km. 1.5 via Milagro-Km26. Milagro 091706, Guayas, Ecuador
* Centro de Biociéncias, Departamento de Micologica, Universidade Federal de Pernambuco,
Avenida da Engenharia, s/n Cidade Universitaria, Recife, PE, 50.740-600, Brazil
‘Instituto de Investigaciones Fundamentales en Agricultura (INIFAT),
Tropical ‘Alejandro de Humboldt’, OSDE, Grupo Agricola,
Calle 1 Esq. 2, Santiago de Las Vegas, C. Habana, Cuba, C.P. 17200

* CORRESPONDENCE TO: dasosa@espol.edu.ec

ABSTRACT—A new species Bactrodesmium pulcherrimum, found on decaying wood
in Ecuador, is described and illustrated. The fungus is distinguished by subnapiform to
broadly subturbinate (maize kernel-shaped) 1-septate asymmetrical ochreous-brown
conidia. Illustrations of Bactrodesmium novageronense and B. simile and a comparison
table of bicellular Bactrodesmium species are also provided.

KEY worDs—asexual fungi, freshwater, hyphomycetes, taxonomy, tropics

Introduction

Saprobic dematiaceous hyphomycetes are highly diverse on plant
materials in Central and South American tropical forests, where many
new genera and species have recently been discovered (Castafieda-Ruiz &

628 ... Espinoza & al.

Fic. 1. Cacao plantation, Guayas Province, Ecuador.

Bactrodesmium pulcherrimum sp. nov. (Ecuador) ... 629

al. 2016). During a survey of hyphomycetes associated with plant debris of
cacao plantations in the Naranjal, Guayas province, southern Ecuador (Fic.
1), we collected a Bactrodesmium specimen that differs remarkably from all
previously described species (Hernandez-Restrepo & al. 2013, Arias & al.
2016). It is described here as a new species, Bactrodesmium pulcherrimum.

Materials & methods

Samples of decaying plant materials were collected and placed in plastic bags for
transport to the laboratory, where they were washed, treated according to Castaneda-
Ruiz & al. (2016), and placed in humid chambers. Several attempts to obtain this
species in pure culture were unsuccessful after using a flamed needle to transfer
conidia to corn meal agar mixed 1:1 with carrot extract and incubating at 25 °C.
Mounts were prepared in PVL (polyvinyl alcohol, lactic acid) and measurements
were made at 1000x magnification. Microphotographs were obtained with an
Olympus BX51 microscope equipped with bright field and Nomarski interference
optics. The type specimen is deposited in the Herbarium of Universidade Federal de
Pernambuco, Recife, Brazil (URM).

Taxonomy

Bactrodesmium pulcherrimum RF. Castafieda, F. Espinoza & D. Sosa,
sp. nov. Pre;
MB 830569

Differs from Bactrodesmium simile by its hemispherical conidial basal cells and maize
kernel-shaped conidia.

Type: Ecuador, Guayas Province, Guayaquil, Naranjal, 02°41’26”S 79°36’46”W, on
decaying wood of Theobroma cacao L. (Malvaceae), 7 March 2018, E. Espinoza & M.
Vera (Holotype, URM 91287).

Erymo oey: Latin, pulcherrimum, meaning most beautiful.

CONIDIOMATA on the natural substrate sporodochial, scattered, pulvinate
ochreous to brown, <20 um diam. Mycelium superficial and immersed composed
of septate, branched, hyaline, smooth hyphae, 2-3 um diam. CONIDIOPHORES
macronematous, mononematous, unbranched or with short lateral branches
scorpioid after 1-2 sympodial elongations of the conidiogenous cells, erect,
flexuous, 2-3-septate, hyaline, smooth, 30-80 x 4-5 um. CONIDIOGENOUS
CELLS monoblastic, integrated or discrete, cylindrical or slightly clavate,
sometimes inflated near the conidiogenous loci, determinate or indeterminate
with sympodial extensions, hyaline, 10-18 x 4-7 um. Conidial secession
rhexolytic. Conrp1A solitary, acrogenous, sometimes acropleurogenous,
broadly obovoid to subnapiform, 1-septate, ochreous-brown, smooth, 18-24 x

630 ... Espinoza & al.

Fic. 2. Bactrodesmium pulcherrimum (holotype, URM 91287): A, B. Conidia with a basal
frill after rhexolytic conidial secession and a detached conidium with a lateral extension of
the conidiogenous cell; C. Conidiogenous cells with lateral sympodial extensions, attached
conidia; D. Detached conidium and conidiogenous loci indicated by arrows; E. Conidiophores,
conidiogenous cells, and conidia; F. Sporodochium. Scale bars: A-E = 10 um; F = 20 um.

Bactrodesmium pulcherrimum sp. nov. (Ecuador) ... 631

13-19 um, asymmetrical, with a basal cell hemispherical, 5-7 x 10-12 um, and
an apical cell semi-elliptical or subglobose, 12-17 x13-17 um, with markedly

pale ochreous-brown lumina.

Fic. 3. Bactrodesmium simile (holotype, XAL CB1689): A. Conidia with a basal frill after
rhexolytic conidial secession. Bactrodesmium novageronense (holotype, INIFAT C84/101-2):
B. Conidia with a basal frill after rhexolytic conidial secession. Scale bars = 10 um.

Note: Bactrodesmium simile Arias & al. (Fic. 3A, TABLE 1) is similar to
B. pulcherrimum in the conidial sizes, but B. simile has broadly pyriform,
obovoid, golden brown to pale olivaceous-brown conidia, with a basal

TABLE 1. Comparison of the 1-septate Bactrodesmium species.

SPECIES

B. novageronense

B. pulcherrimum

B. simile

COLOR

Pale brown

Ochreous-brown

Golden brown to
pale olivaceous
brown

CONIDIAL MORPHOLOGY

SHAPE & SIZE
(um)
Obovoid
to subglobose
8-13 x 7-10

Broadly obovoid
to subnapiform
18-24 x 13-19

Broadly pyriform
to obovoid
19-24 x 12-16

BASAL CELL
(um)
Hemispherical
3-3.5 x 5-7

Hemispherical
5-7 x 10-12

Cuneiform or
obconical
7.5-11 x 5-8.5

REFERENCE

Castafieda-Ruiz
1985

This paper

Arias & al.
2016

632 ... Espinoza & al.

cell cuneiform, 7.5-11 x 5-8.5 um (Arias & al. 2016). Bactrodesmium
novageronense R.F. Castaneda (Fic. 3B, TABLE 1) is similar to B. pulcherrimum
but can be distinguished by its smaller conidia (8-13 x 7-10 um;
Castanieda-Ruiz 1985).

Acknowledgments

We are indebted to Dr. Josiane Santana Monteiro (Museu Paraense Emilio
Goeldi, Belém, Brazil) and Dr. De-Wei Li (The Connecticut Agricultural
Experiment Station Valley Laboratory, Windsor, U.S.A.) for their critical reviews.
The authors thank Mr. Jairo Gonzalez for providing permission to collect samples.
The authors are grateful to Escuela Superior del Litoral (ESPOL), CIBE, for financial
support and the International Society for Fungal Conservation for facilities. RFCR
is grateful to the Cuban Ministry of Agriculture. We acknowledge the websites
provided by Dr. P.M. Kirk (Index Fungorum) and Dr. K. Bensch (MycoBank).
Dr. Lorelei Norvell’s editorial review and Dr. Shaun Pennycook’s nomenclature
review are greatly appreciated.

Literature cited

Arias RM, Heredia G, Castafieda-Ruiz RF. 2016. Two new species of Bactrodesmium and
Dictyoaquaphila from Mexico. Mycotaxon. 131: 291-295. https://doi.org/10.5248/131.291

Castafeda-Ruiz RE 1985. Deuteromycotina de Cuba. Hyphomycetes 2. Instituto de
Investigaciones Fundamentales en Agricultura Tropical, Cuba. 23 p.

Castafieda-Ruiz RF, Heredia G, Gusmao LFP, Li DW. 2016. Fungal diversity of Central and
South America. 197-217, in: DW Li (ed.). Biology of Microfungi. Springer International
Publishing. https://doi.org/10.1007/978-3-319-29137-6_9

Hernandez-Restrepo M, Mena-Portales J, Gené J, Cano J, Guarro J. 2013. New
Bactrodesmiastrum and Bactrodesmium from decaying wood in Spain. Mycologia 105:
172-180. https://doi.org/10.3852/12-004

MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 633-635
https://doi.org/10.5248/134.633

Dendrographium multiseptatum sp. nov.
from China

Li-Guo Ma, YUE-LI ZHANG, Bo ZHANG,
Kal QI, CHANG-SONG LI, JUN-SHAN QT

Shandong Key Laboratory of Plant Virology, Institute of Plant Protection,
Shandong Academy of Agricultural Sciences, Jinan, Shandong, 250100, China

“CORRESPONDENCE TO: gi999@163.com

ABSTRACT—A new species, Dendrographium multiseptatum, is described and illustrated from
specimens collected on rotten twigs in China. This fungus is characterized by synnematous
conidiophores with polytretic conidiogenous cells producing obclavate, 6-9-distoseptate,
pale brown conidia.

KEY wWoRDS—asexual morph, microfungi, hyphomycete, taxonomy

Introduction

Dendrographium Massee was established by Massee (1892) to
accommodate the Brazilian type species D. atrum Massee. The genus is
characterized by synnematous, macronematous, unbranched conidiophores
with polytretic, integrated, terminal (becoming intercalary), sympodial,
cylindrical conidiogenous cells that produce solitary, acropleurogenous,
simple, distoseptate conidia (Ellis 1971, Seifert & al. 2011). Species
assignment in Dendrographium is based primarily on conidial morphology,
including shape, septal number, size, pigmentation, and ornamentation. Six
additional species have been described (all from India), but Dendrographium
kamatii V.G. Rao with monotretic conidiogenous cells has been recombined
as Corynespora kamatii (V.G. Rao) M.B. Ellis (Rao 1963, Ellis 1976). Ghosh
& al. (1977) provided a comparison table for the six Dendrographium species
(including D. kamatii) reported from India.

634 ... Ma &al.

During a survey on wood-inhabiting microfungi in China, a new
Dendrographium species was found. As this species did not match any of the
currently described species in the genus, it is proposed here as new. Specimens
are deposited in the Herbarium of Institute of Plant Protection, Shandong
Academy of Agricultural Sciences, Jinan, Shandong, China (HSAAS) and
the Mycological Herbarium, Institute of Microbiology, Chinese Academy of
Sciences, Beijing, China (HMAS).

Taxonomy

Dendrographium multiseptatum L.G. Ma & J.S. Qi, sp. nov. FIG. 1
MB 832847

Differs from Dendrographium atrum by its narrower conidiophores and its shorter,
broader conidia with fewer distosepta and from D. mitteri by its longer synnemata, and
solitary, shorter, 6-9-distoseptate conidia.

Type: China, Shandong Province, Taian City, Taishan District, on rotten twigs of an
unidentified plant, 22 May 2018, L.G. Ma (Holotype, HSAAS 0415; isotype, HMAS
146095).

ETyMOLoGy: multiseptatum, referring to the numerous conidial septa.

CoLoNnIEs on the natural substratum effuse, black to dark brown, tufted.
Mycelium mostly immersed in the substratum. Synnemata large, black,
erect, tapered towards the apex, with dark brown stalks, consisting of
parallel conidiophores, 520-1380 um long, 25-85 um diam. at the base.
CONIDIOPHORES macronematous, synnematous, mostly adpressed along
lower part of the length, divergent towards the upper part, unbranched,
erect, straight or flexuous, cylindrical, septate, dark brown, smooth,
thick-walled, brown towards the apex, <1400 um long, 3.5-6.5 um diam.
CONIDIOGENOUS CELLS integrated, terminal becoming intercalary,
polytretic, cylindrical, brown, smooth, thick-walled, 8-24 x 5.0-7.5 um.
Conipi1A solitary, acropleurogenous, simple, obclavate, 6—9-distoseptate,
smooth, thick-walled, pale brown, 32-66 x 8-11.5 um, tapered towards the
apex, with a basal scar, truncate at the base, 2.5-3.5 um diam.

CoMMENTS—Dendrographium multiseptatum most closely resembles
D. atrum and D. mitteri Syd. in conidial shape. However, D. atrum has
broader (6-10 um) conidiophores and longer and narrower conidia
with more distosepta (40-130 x 7-9 um, 6-15 distosepta; Massee 1892).
Dendrographium mitteri differs by its shorter synnemata (170-250 um) and
occasionally catenate, longer (35-110 um), 3-10-septate conidia (Sydow &
Mitter 1933).

Dendrographium multiseptatum sp. nov. (China) ... 635

Fic. 1. Dendrographium multiseptatum (holotype, HSAAS 0415): A-D. Synnematous
conidiophores, conidiogenous cells, and conidia; E. Synnematous conidiophores with
conidiogenous cells and conidium; F. Conidia.

Acknowledgments

The authors express gratitude to Dr. De-Wei Li (The Connecticut Agricultural
Experiment Station Valley Laboratory, Windsor, U.S.A.) and Dr. Ze-Fen Yu
(Laboratory for Conservation and Utilization of Bio-resources, Yunnan University,
Kunming, P.R. China) for serving as pre-submission reviewers and to Dr. Shaun
Pennycook for nomenclatural review. This project was supported by Young Talents
Training Program of Shandong Academy of Agricultural Sciences (CXGC2018E04),
National Key R & D Program of China (2016YFD0300700, 2017YFD0201700), and
National Natural Science Foundation of China (31400019).

Literature cited

Ellis MB. 1971. Dematiaceous hyphomycetes. Commonwealth Mycological Institute, Kew,
Surrey, England.

Ellis MB. 1976. More dematiaceous hyphomycetes. Commonwealth Mycological Institute,
Kew, Surrey, England.

Ghosh RN, Pathak NC, Singh MS. 1977. Two new records of stilbaceous fungi from India.
Norwegian Journal of Botany 24: 79-81.

Massee GE. 1892. Notes on exotic fungi in the Royal Herbarium, Kew. Grevillea 21: 1-6.

Rao VG. 1963. A new species of Dendrographium from India. Current Science 32: 473-474.

Seifert KA, Morgan-Jones G, Gams W, Kendrick B. 2011. The genera of Hyphomycetes.
CBS Biodiversity Series 9. 997 p.

Sydow H, Mitter JH. 1933. Fungi indici. I. Annales Mycologici 31: 84-97.

MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 637-641
https://doi.org/10.5248/134.637

Haematomma pluriseptatum sp. nov.
from China

CONGCONG MIAO *!, RONG TANG **4,
LINLIN DoNG?, ZHAOJIE REN?, ZUNTIAN ZHAO **

"Key Laboratory of Plant Stress Research, College of Life Sciences &
? Institute of Environment and Ecology:

Shandong Normal University, Jinan, 250014, PR. China
* Shandong Museum, Jinan, 250014, P. R. China

‘The First High School of Liangshan, Shandong Province, 272600, P. R. China

“CORRESPONDENCE TO: ccmjy123@163.com

ABSTRACT—A new species, Haematomma pluriseptatum, is described from southwestern

China. This corticolous species is characterized by large ascospores with (19-)20-26(-27)

septa and by the presence of atranorin, russulone, and pseudoplacodiolic acid. A detailed

description of its morphology, high resolution photographs, chemistry, comments, and

distribution are provided. Related lichen taxa are discussed, and a key to the species of

Haematomma from China is also provided.

Key worps—East Asia, Haematommataceae, Lecanorales, lichenized fungi, taxonomy

Introduction

The crustose Haematomma lichens, with their small but brilliant-red

lecanorine apothecia, are often conspicuous on smooth-barked trees in

warm-temperate to tropical regions of the world (Staiger & Kalb 1995,

Brodo & al. 2008). Haematomma was proposed originally to house two

species, H. vulgare A. Massal. and H. ventosum (L.) A. Massal. (Massalongo

1852). The genus is characterized by Haematomma-type asci, blood-red

lecanorine apothecia, and transversely septate, submuriform to muriform

*CONGCONG MIAO & RonG TANG contributed equally to this work.

638 ... Miao, Tang & al.

ascospores (Brodo & al. 2001, Elix 2004, Nelsen & al. 2006, Nash & al. 2004,
Smith & al. 2009). Apothecial pigmentation and secondary chemistry play
important roles for species delimitation in this genus (Messuti & de la Rosa
2009). The number of spore septa is also used as an important character
in separating species (Nelsen & al. 2006). Of the forty Haematomma
species reported in the world, twelve have been reported in China (Brodo
& al. 2001, Elix 2004, Nelsen & al. 2006, Nash & al. 2004, Smith & al.
2009, Tang & al. 2018). For this study, we examined about two hundred
specimens of Haematomma morphologically and chemically, describe a
new species, H. pluriseptatum, from China, and present a key to the species
of Haematomma from China.

Materials & methods

Four collections are deposited in the Lichen Section of the Botanical Herbarium,
Shandong Normal University, Jinan, China (SDNU), and one is deposited in the
Lichen Herbarium of the Kunming Institute of Botany, Kunming, China (KUN).
The specimens were examined morphologically and anatomically morphological
under an Olympus SZ51 stereomicroscope and Olympus CX21 polarizing
microscope. Both thallus and medulla were tested with K (a 10% KOH aqueous
solution) and C (a saturated aqueous NaOCl solution). The lichen substances were
identified using standardized thin layer chromatography techniques (TLC) with
solvent system A and C (Orange & al. 2010). The specimens were photographed
using a DP72 camera on Olympus SZX16 and BX61 microscopes.

Taxonomy

Haematomma pluriseptatum R. Tang, sp. nov. Fie. 1
MB 830618

Differs from Haematomma rufidulum by its ascospores with more septa and its
production of pseudoplacodiolic acid.

Type: China. Yunnan, Diging Co., Shangri-La, Tianbao Mt., 27°36’37”N 99°53’37”E,
alt. 3790 m, on bark of living trees, 17 Aug. 2018, Wang Chunxiao 20181135 (Holotype,
SDNU).

EryMo.ocy: The epithet pluriseptatum refers to the morphology of the ascospores with

many septa.
Thallus crustose, corticolous, pale greyish to greenish grey, rugose rimose-
areolate to areolate, continuous, thickness 0.6 mm. Soredia and isidia
absent. Apothecia scattered, 0.3-0.95(-1.0) mm diam., sessile, convex,
constricted at the base, smooth when young, red. Disc orange-yellow to
brownish yellow, white pruina present when young but absent when mature;
Thalline margin well developed; amphithecium present, 70-90 um thick.

Haematomma pluriseptatum sp. nov. (China) ... 639

Fic. 1. Haematomma pluriseptatum (holotype, SDNU (Wang Chunxiao 20181135)). A. Thallus
and apothecia; B. Apothecium section; C. K+ reaction (red) of epihymenium and outer exciple;
D. Ascus; E. Ascospores. Scale bars: A = 200 um; B, C = 100 um; D = 5 um; E = 10 um.

Epihymenium orange, K+ red, 9-20 um thick. Hymenium hyaline, 70-95
um high. Hypothecium hyaline or slightly brown. Paraphyses branched
and anastomosing. Asci clavate, containing 8 spores. Ascospores twisted in
ascus, filiform, straight or slightly curved, (19-)20-26(-27)-septate, 70-95 x
5-6.5(—7) um. Conidia absent.

CHEMISTRY—Cortex K+ yellow, C-, KC-. Atranorin, russulone, and
pseudoplacodiolic acid detected by TLC.

ADDITIONAL SPECIMENS EXAMINED: CHINA. YUNNAN, Diging Co., Shangri-La,
Tianbao Mt., 27°36’37”N 99°53’37’E, alt. 3790 m, on bark of living trees, 17 Aug.
2018, Wang Chunxiao 20181175, 20181209, 20181126 (SDNU); Deqin Co., Yubeng
Village, Xiaonong Base Camp. 28°23.92’N 98°46.16’E, alt. 3500 m, on bark of living
trees, 14 Sep. 2012, Niu Dongling 12-36322 (KUN-L).

COMMENTS—Haematomma pluriseptatum is characterized by the large
number of septa (20-27) in each ascospore and the presence of atranorin,
russulone and pseudoplacodiolic acid in the thallus.

640 ... Miao, Tang & al.

The new ascomycete is morphologically similar to H. rufidulum (Fée)
A. Massal., which differs by the occurrence of slightly irregular, white,
convex to hemispherical soredia and elongate-fusiform, 8-25-celled,
50-72(-85) x (3.5—)4-6(-—7) um ascospores (Brodo & al. 2008). Secondary
metabolites also differ: H. pluriseptatum contains pseudoplacodiolic acid,
while H. rufidulum contains placodiolic acid.

Haematomma pluriseptatum may also be confused with H. dolichosporum
(B. de Lesd.) Kalb & Staiger, which also produces twisted spores in the
asci, but H. dolichosporum is readily distinguished by its shorter (42-62
um) spores with fewer (7—9(-11)) septa and the production of placodiolic
acid (Staiger & Kalb 1995; Brodo & al. 2008).

Key to the species of Haematomma in China

1. Epihymenium with russulone, K+ Ted 25 soe sien seston nie Sn olew slew ales 2
1. Epihymenium with haematommone, K+ purple (entirely dissipating) ........... 8
2 siallastcomtaimi ne UgMte BOR | nse ne hms nadie Rate ree Sad een Bade ea ade eek ae H. fauriei
2. Uhalluslackine-usnic acid.) 25. ebay. Bey ee tie ee tk el MEE in ee ae De geee ts mes 3
SeOMTOCKE 5h nds Awe ade eH eH lng W eiagee nN efegeed ehogee me Ong mee H. fenzlianum
Pe EU Dial Raters, onye- dag, nyee apa > aeons teen otis #o genie key ie sn gh mesa ean eM S ees ote ee -:
4, Thallus containing placodiolic acid ............. 0... e eee eee eee H. rufidulum
#. balls ekino-placOd iol Ge: dGid. cna ee Ms aren neers Basten Paks ea ede oe Sly or Rodgers Bootes 5
>) thallus lacking sphderophotin ts, atac. eitore a ara eee nea ee hee Paes Pee 5 6
5. Thallus containing sphaerophorin,

Sporesto= IA seclledanw to, We M rim oe meh tans Rens Rees CAR a eRe ne ee 7
6. Thallus containing pseudoplacodiolic acid,

spores’ 20=27—celled oo n.cey vin eee Reed ee oad Bee EE Be oeS H. pluriseptatum
6. Thallus lacking pseudoplacodiolic acid,

spores S=l6scelled) .. is. ese eee ae ee eee es H. puniceum subsp. pacificum
Fc SPOVesOa7 ACE 6 x 4 bsg kag t rhea y eAGY 4S MENS MEE EAR H en tie Dy nae H. persoonii
7 Spores 01a xcelled. 28 Anh thal fatter Ma nat Ane nak Met May H. collatum
SeSPOTEs MOE SUDEIUTIIOLI, 5 oF 58 ue «Aste eg. ike recom ga-rhc om gro mG aaa oni arec phe e cn eget wie 9
SPS Pores Su DHMITMOLN a ac, tila kn eas Cees earn ies dee tten aeeeeen Rete ss hae H. wattii
Re ApOecia: Sorediatee.. M.S ath. a sgeh ese h ead ven tak wonton moe ete H. caperaticum
9 Apothecranorsorediate +5. 2565 28 ls oh Besos eed bs ee hee ema ee et Metgs oe eins 10
10. Thallus containing isoplacodiolic, isopseudoplacodiolic acids ...... H. flexuosum
10. Thallus lacking isoplacodiolic, isopseudoplacodiolic acids ................... 11
Ines poreg celled 7 itr eh Fut i oP RNa aI H. accolens

I Sporess eas ceed ie is carte Merde Bevery Rebstes Roden Sods ee Babee hon H. africanum

Haematomma pluriseptatum sp. nov. (China) ... 641

Acknowledgments

We thank Dr. Klaus Kalb (Lichenological Institute Neumarkt, Germany) and
Dr. Shou-Yu Guo (State Key Laboratory of Mycology, Institute of Microbiology,
Chinese Academy of Sciences, Beijing) for presubmission review. The authors would
also like to thank Lisong Wang and Xinyu Wang (Kunming Institute of Botany, CAS,
China) for assistance during this study. This work was supported by the National
Natural Science Foundation of China Youth Science Foundation (31600100), and
Emergency management project of National Natural Science Foundation of China
(31750001).

Literature cited

Brodo IM, Sharnoff SD, Sharnoff S. 2001. Lichens of North America. Yale University Press, New
Haven and London, 795 p.

Brodo IM, Culberson WL, Culberson CE. 2008. Haematomma (Lecanoraceae) in North and
Central America. Bryologist 111: 363-423.
https://doi.org/10.1639/0007—2745(2008) 111[363:hlinac]2.0.co;2

Elix JA. 2004. Haematommataceae. 4-10, in: Flora of Australia 56A—Lichens 4. Australian
Biological Resources Study/CSIRO Publishing. 222 p.

Massalongo AB. 1852. Ricerche sull’ autonomia dei licheni crostosi. Verona: Friziero. 207 p.

Messuti MI, de la Rosa IN. 2009. Notes on the genus Haematomma (Ascomycota, Lecanoraceae)
in Argentina. Darwiniana 47(2): 297-308.

Nash TH, Ryan BD, Diederich P, Gries C, Bungartz F. 2004. Lichen flora of the Greater Sonoran
Desert Region, vol. 2. Tempe, Lichens Unlimited.

Nelsen MP, Liicking R, Chaves JL, Sipman HJM, Umafa L, Navarro E. 2006. A first assessment
of the Ticolichen biodiversity inventory in Costa Rica: the genus Haematomma (Lecanorales:
Lecanoraceae). Lichenologist 38: 251-262. https://doi.org/10.1017/s0024282906005573

Orange A, James PW, White FJ. 2010. Microchemical methods for the identification of lichens.
2™ edition. London, British Lichen Society.

Smith CW, Aptroot A, Coppins BJ, Fletcher A, Gilbert OL, James PW, Wolseley PA. 2009.
The lichens of Great Britain and Ireland. London, British Lichen Society.

Staiger B, Kalb K. 1995. Haematomma-Studien: I. Die Flechtengattung Haematomma. Bibliotheca
Lichenologica 59. 198 p.

Tang R, Yan SK, Sun MJ, Zhang LL. 2018. New records of Haematomma and Ophioparma from
China. Mycotaxon 133(1): 175-181. https://doi-org/10.5248/133.175

MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 643-648
https://doi.org/10.5248/134.643

New records of Didymium inconspicuum,
D. karstensii, and D. rugulosporum
from China

CHAOFENG YUAN, SHU LI, WAN WANG,
SHUWEI WEI, Qi WANG, YU LI
Engineering Research Center of Chinese Ministry of Education

for Edible and Medicinal Fungi, Jilin Agricultural University,
Changchun, Jilin 130118 China

* CORRESPONDENCE TO: giwang@jiau.edu.cn

ABSTRACT—Didymium inconspicuum, D. karstensii, and D. rugulosporum are reported as
new records for China. Morphological characteristics were observed and described by light
microscopy and scanning electron microscopy and are discussed and compared with similar
species.

Key worps—Didymiaceae, Myxogastrea, taxonomy

Introduction

Didymium was introduced by Schrader (Schrader 1797) and characterized

by sporophores with a peridium covered with stellate lime crystals. In

contrast, the capillitium is almost always limeless. The lime crystal nature on

the peridium separates Didymium from Diderma, in which the lime occurs

as amorphous granules (Martin & Alexopoulos 1969). Currently Didymium

includes approximately 94 species around the world (Kirk & al. 2008, Lado
2018), of which 35 have been recorded in China (Li & al. 1996, Liu & Chen
1998, Chen 1999, Li 2008, Liu & Chang 2011, Gao & al. 2018). The objectives
of this work were to examine some putative Didymium samples, characterize

them by light and scanning electron microscopy, and compare them with

similar species.

644 ... Yuan & al.

Materials & methods

Specimen collection and moist chamber culture

Didymium rugulosporum was collected from Huzhong Nature Reserve,
Heilongjiang Province in 2016. Didymium karstensii was collected from Changbai
Mountain, Jilin Province in 2018. Didymium inconspicuum was collected from the
desert transition zone of Suwu Township, Mingin County, Gansu Province in 2016.
The specimens are deposited in the Herbarium of the Mycological Institute of Jilin
Agricultural University, Changchun, China (HMJAU).

Morphological identification

The morphological characteristics of the sporocarps were observed by light
microscope and scanning electron microscope. Each sporocarp was placed on a
slide and one drop of 4% KOH added. The slide cover was placed on top and gently
tapped, and filter paper used to remove excess solution. The capillitium, calcareous
crystals, and spores were observed under a Leica DM 2000 light microscope
(LM). Up to 20 spores were randomly selected and their diameters measured.
The ultrastructure was observed under the Hitachi SU8010 scanning electron
microscope (SEM) operating at 5kV (Martin & Alexopoulos 1969, Zhu & al. 2012,
Li & al. 2017).

Taxonomy

Didymium inconspicuum Nann.-Bremek. & D.W. Mitch.,
Proc. Kon. Ned. Akad. Wetensch., C 92: 508. 1989 Fic. 1

Sporocarps sessile, scattered, occasionally mingled with — short
plasmodiocarps, small and inconspicuous. 0.5-1 mm long, about 0.5 mm high,
ovoid, often depressed in the center, grayish white. Columella absent. Peridium
membranous, thin, coated with grayish white lime crystals. Capillitium profuse,
threads slender and curved, pale brown, 1-1.5 um diam., often branching and
anastomosing to an incomplete network, sometimes with flattened and darker
colored sections on the branches. Spores dark brown in mass, brown by LM,
ovoid to globose, 8-11 um diam., evenly slightly warted.

SPECIMEN EXAMINED: CHINA, GANSU PROVINCE, Mingin County, Suwu Township,

38°16'42”N 103°06’17’E, 1360 m, on leaf by moist chamber culture, 16 September 2016,

Chaofeng Yuan & Shuwei Wei (HMJAU 10650).
CoMMENTs: Didymium inconspicuum is similar to Didymium obducens
P. Karst. [= D. fulvum Sturgis] and Didymium ochroideum G. Lister. The spore
of D. obducens is tuberculate on one side, while the other side is smooth and
the color is pale, but the spore of D. inconspicuum has relatively uniform
small warts. The spore of D. ochroideum is 6-8 um diam. whereas the spore
of D. inconspicuum is 8-11 1m diam.

Didymium spp. new to China... 645

Fic 1. Didymium inconspicuum. A- C. Sporocarps; D. Capillitium (LM); E. Spores (LM);
F. Capillitium (SEM); G. Lime crystal (SEM); H. Spores (SEM). Scale bars: A- C = 500 um;
D, F= 10 pm; E, G, H=5 pm.

Nannenga-Bremekamp (1989) described D. inconspicuum as having white
or pale yellow-brown tiny lime crystals, with a coarse capillitium and spores
that are 12-14(-15) um diam.; Schnittler & Novozhilov (2000) described this
species as having 3-6 um long lime crystals smaller than spores, a pale to pale
brown, 1-2(-2.5) um diam. capillitium, and (9.5—)10.5-11.5(-12.5) um diam.
spores. Our specimen has white lime crystals about 5-8 um diam., a light
brown 1-1.5 um diam. capillitium, and spores 8-11 um diam.; other features
are consistent with the published descriptions.

Didymium karstensii Nann.-Bremek., Acta Bot. Neerl. 13: 247. 1964 FIG. 2

Sporocarps gregarious, sessile or with a short stalk, white, subglobose,
1 mm wide, up to 0.5 mm high, stalk (when present) calcareous, yellowish
white, about 0.2 mm long. Peridium double, outer layer white, with stellate
lime crystals about 5 um diam. forming the wrinkled shell; the inner layer
membranous and colorless. Columella white, calcareous, slightly thickened at

646 ... Yuan & al.

Fic 2. Didymium karstensii. A. Sporocarps; B. Capillitium and swelling (LM); C. Spores
(LM); D. Lime crystals (SEM); E. Capillitium (SEM); F. Spores (SEM). Scale bars: A = 1 mm;
B-E = 10 um; F=5 um.

the base. Capillitium pale brown, threads slender, branching and anastomosing
to a network, with dark swellings, 2-3 um diam., attached to the peridium and
to the base of the sporocarp. Spores dark brown in mass, brown by LM, densely
warted and ridged, 7.5-10 um diam.

SPECIMEN EXAMINED: CHINA, JILIN PROVINCE, Changbai Mountain, 42°22’18”N

128°00'14”E, 828 m, on the leaf, 5 September 2018, Chaofeng Yuan & Shuwei Wei

(HMJAU 10651).
Comments: Didymium karstensii is similar to Didymium squamulosum (Alb. &
Schwein.) Fr., but D. karstensii is inconspicuously umbilicate at the base, whereas
D. squamulosum is deeply umbilicate at the base. Nannenga-Bremekamp
(1964) described stalks of D. karstensii as generally not exceeding half the
diameter of the sporocarps, and spinulose spores, 10-12 um diam. Demirel &
Kasik (2012) described stalks of D. karstensii generally not exceeding 0.2 mm
long, and warted spores, 10-12 um diam. Our specimen had stalks about 0.2
mm long and warted spores, 7.5-10 um across; other features are consistent
with the published descriptions.

Didymium rugulosporum Kowalski, Mycologia 61: 636. 1969 Fic. 3

Sporocarps sessile, scattered to clustered, globose to subglobose, 1-2.5 mm
diam., white. Peridium double, the outer layer thick and fragile, white smooth
calcareous shell, stellate lime crystals; the inner layer membranous, thin,
colorless and transparent. Columella absent. Capillitium dense, rigid, brown,

Didymium spp. new to China ... 647

Fic 3. Didymium rugulosporum. A, B. Sporocarps; C. Capillitium and spores (LM); D. Capillitium
and peridium (SEM); E. Lime crystal (SEM); FE. Spores (SEM). Scale bars: A, B = 1 mm; C = 20 um;
D = 10 um; E, F=5 um.

1-1.5 um diam., weakly attached to the base and apex of the sporocarps, brown,
branched with many colorless swellings. Spores dark brown in mass, purple
brown by LM, globose, 17-19 um diam., with large dense warts about 1.5 um
high and with some ridges forming a reticulum.
SPECIMEN EXAMINED: CHINA, HEILONGJIANG PROVINCE, Huzhong Nature Reserve,
52°06'02”N 123°20'38”E, 168 m, on the bark, 24 September 2016, Wan Wang (HMJAU
10652).
Comments: Didymium rugulosporum is similar to Didymium trachysporum
G. Lister and Didymium quitense (Pat.) Torrend, which differ by their smaller
spores (D. trachysporum 9-10 um diam.; D. quitense is 12-15 um diameter).
Kowalski (1969) described spores of D. rugulosporum as (16—)18-20(-22) um
diam. Our specimen had spores 17-19 um diam.; other characteristics are
consistent with the original description.

Acknowledgments

We would like to thank Tom Hsiang (University of Guelph, Canada) and Paul
M. Kirk (RBG Kew, UK) for presubmission reviews of this manuscript. This work
was financed by the National Natural Science Foundation of China (NO.31770011,
NO.31370065).

Literature cited

Chen SL. 1999. Fungal flora of tropical Guangxi, China: a survey of myxomycetes from southwestern
Guangxi. Mycotaxon 72: 393-401.

648 ... Yuan & al.

Demirel G, Kasik G. 2012. Four new records for Physarales from Turkey. Turkish Journal of
Botany 36: 95-100. https://doi.org/10.3906/bot-1010-12

Gao Y, Yan SZ, Wang GW, Chen SL. 2018. Two new species and two new records of myxomycetes
from subtropical forests in China. Phytotaxa 350(1): 51-63.
https://doi.org/10.11646/phytotaxa.350.1.6

Kirk PM, Cannon PF, Minter DW, Stalpers JA. 2008. Ainsworth & Bisby’s dictionary of the
fungi. 10th edition. CAB International Publishing, Wallingford. 771 p.

Kowalski DT. 1969. A new coprophillous species of Didymium. Mycologia 61(3): 635-639.
https://doi.org/10.2307/3757253

Lado C. (2018). An online nomenclatural information system of Eumycetozoa. Real Jardin
Botanico, CSIC. Madrid, Spain. http://www.nomen.eumycetozoa.com (Accessed:
September 2018).

Li Y. 2008. Flora myxomycetes sinicorum. Science Press.

Li HZ, Li Y, Wang Q. 1996 [“1995-96”]. Myxomycetes from China XIII: a new species of
Didymium. Mycosystema 8-9: 173-175.

Li S, Wang W, Wang W, Wang Q, Li Y. 2017. Morphology and life-cycle of Fuligo leviderma,
a newly recorded myxomycete species of China. Mycosystema 36(4): 528-531.
https://doi.org/10.13346/j.mycosystema.160122

Liu CH, Chang JH. 2011. Myxomycetes of Taiwan XXIII. The genera Diachea and Didymium.
Taiwania 56: 287-294. https://doi.org/10.6165/tai.2011.56(4).287

Liu CH, Chen YE. 1998. Myxomycetes of Taiwan X. Three new records of Didymium. Taiwania
43: 177-184. https://doi.org/10.6165/tai.1998.43(3).177

Martin GW, Alexopoulos CJ. 1969. The myxomycetes. University of Iowa Press, Iowa City.
560 p.

Nannenga-Bremekamp NE. 1964. Notes on myxomycetes VIII. A new species of
Didymium from the Netherlands. Acta Botanica Neerlandica 13: 246-249.
https://doi.org/10.1111/j.1438-8677.1964.tb00155.x

Nannenga-Bremekamp NE. 1989. Notes on myxomycetes XXIII. Seven new species of
myxomycetes. Proceedings, Koninklijke Nederlandse Akademie van Wetenschappen,
Series C, 92: 505-515.

Schnittler M, Novozhilov YK. 2000. Myxomycetes of the winter-cold desert in western
Kazakhstan. Mycotaxon 74(2): 267-285.

Schrader HA. 1797. Nova genera plantarum. Pars prima, Lipsiae.

Zhu H, Song XX, Li $, Wang Q, Li Y. 2012. Three new recorded species of myxomycetes in
China. Mycosystema 31(6): 947-951. https://doi.org/10.13346/j.mycosystema.2012.06.021

MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 649-662
https://doi.org/10.5248/134.649

Rhomboidia wuliangshanensis gen. & sp. nov.
from southwestern China

TaI-MIN XU?”, XIANG-Fu Liu3, YuU-HuI CHEN’, CHANG-LIN ZHAO?

"Yunnan Provincial Innovation Team on Kapok Fiber Industrial Plantation;
? College of Life Sciences; * College of Biodiversity Conservation:
Southwest Forestry University, Kunming 650224, PR. China

" CORRESPONDENCE TO: fungichanglinz@163.com

ABSTRACT—A new, white-rot, poroid, wood-inhabiting fungal genus, Rhomboidia,
typified by R. wuliangshanensis, is proposed based on morphological and molecular
evidence. Collected from subtropical Yunnan Province in southwest China, Rhomboidia is
characterized by annual, stipitate basidiomes with rhomboid pileus, a monomitic hyphal
system with thick-walled generative hyphae bearing clamp connections, and broadly
ellipsoid basidiospores with thin, hyaline, smooth walls. Phylogenetic analyses of ITS and
LSU nuclear RNA gene regions showed that Rhomboidia is in Steccherinaceae and formed
as distinct, monophyletic lineage within a subclade that includes Nigroporus, Trullella, and
Flabellophora.

Key worps—Polyporales, residual polyporoid clade, taxonomy, wood-rotting fungi

Introduction

Polyporales Gaum. is one of the most intensively studied groups of fungi
with many species of interest to fungal ecologists and applied scientists (Justo
& al. 2017). Species of wood-inhabiting fungi in Polyporales are important
as saprobes and pathogens in forest ecosystems and in their application in
biomedical engineering and biodegradation systems (Dai & al. 2009, Levin
& al. 2016). With roughly 1800 described species, Polyporales comprise about
1.5% of all known species of Fungi (Kirk & al. 2008). Currently, there are
46 genomes of polyporalean taxa available from the Joint Genome Institute
MycoCosm portal (Grigoriev & al. 2013).

650 ... Xu & al.

TABLE 1. Species and sequences used in the phylogenetic analyses

SPECIES

Abortiporus biennis
Antrodiella americana

A. semisupina
Antrodiella sp.
Climacocystis borealis
Diplomitoporus flavescens

Elaphroporia ailaoshanensis

Flabellophora sp.
Flaviporus brownii

E liebmannii

Frantisekia mentschulensis

Hypochnicium bombycinum
H. lyndoniae

Irpex lacteus

Ischnoderma benzoinum

I. resinosum

Junghuhnia crustacea

J. micropora

Loweomyces fractipes

Mycorrhaphium adustum

SAMPLE

TFRI 274

Gothenburg 3161

FCUG 960
X 418

KH 13318
X 84
CLZhao 595
CLZhao 596
X340

X 1216

X 251

X 249

X 666

BRNM 710170

1377

MA 15305

NL 041031

CBS 431.48

DO 421/951208

KHL 12099

FD-328

X 1127

X 262
Spirin 2652
X 1149

X 1253

X 1250
8024

Dai 10173

GENBANK ACCESSION NO.

ITS
EU232187
JN710509
EU232182
JN710523
JQ031126
FN907908
MG231568
MG231572
JN710534
JN710537
JN710541

JN710539

JN710540
FJ496728
JN710544
FN552537
JX124704
MH856423
JX109852
JX109841

KP135303

JN710554
JN710553
JN710559
JN710570
JN710569

JN710568

JN710573

KC485537

LSU
EU232235
JN710509
EU232266
JN710523
JQ031126
MG231568
MG231572
JN710534
JN710537
JN710541

JN710539

JN710540

JN710544
JX124704
MH867969
JX109852
JX109841

KP135225

JN710554
JN710553
JN710559
JN710570
JN710569
JN710568
JN710573

KC485554

REFERENCES

Larsson 2007
Miettinen & al. 2012
Binder & al. 2005
Miettinen & al. 2012
Binder & al. 2013
Miettinen & al. 2012
Wu & al. 2018

Wu & al. 2018
Miettinen & al. 2012
Miettinen & al. 2012
Miettinen & al. 2012
Miettinen & al. 2012
Miettinen & al. 2012
TomSsovsky & al. 2010
Miettinen & al. 2012
Binder & al. 2013
Binder & al. 2005
Vu & al. 2019
Binder & al. 2013
Binder & al. 2013

Floudas & Hibbett
2015

Miettinen & al. 2012
Miettinen & al. 2012
Miettinen & al. 2012
Miettinen et al. 2012
Miettinen & al. 2012
Miettinen & al. 2012

Miettinen & al. 2012

Nigroporus vinosus

Panus conchatus
P strigellus

Physisporinus
sanguinolentus

P. vitreus

Podoscypha venustula

Pseudolagarobasidium
acaciicola

P. belizense

Rhomboidia
wuliangshanensis

Skeletocutis
novae-zelandiae

Spongipellis spumeus

Steccherinum fimbriatum

S. ochraceum

Trullella dentipora
T. duracina
T. polyporoides

Xanthoporus syringae

Rhomboidia wuliangshanensis gen. & sp. nov. (China) ... 651

X 839

8182

BHS2008- 100

X 1234

INPA 243940

BRNM 699576

3163

KHL11959

CBS 486.72

CBS 65684

CBS 115543

CBS 115544

CFMR DLC 04-31

CLZhao 4406 [T]

CLZhao 4411

Ryvarden 38641

PRM 891931
BRNM 712630
BRNM 734877
KHL 11905
Ryberg s.n.
KHL 11902

X 200

X 290

X 510

X 339

Cui 2177

Gothenburg 1488

N710576

JN710728
JX109857
JN710579
JQ955725

FJ496671

JN710580

JQ031129

MH860538

JN649367

DQ517883

DQ517882

JQ070173

MK860715

MK860716

JN710582

HQ728287
HQ728288
HQ728283
EU118668
EU118669
JQ031130
JN710512
JN710513
JN710602
JN710606
DQ789395

JN710607

N710576

JN710728
JX109857
JN710579
JQ955732

FJ496725

JN710580

JQ031129

MH872244

JN649367

MK860710

MK860711

JN710582

HQ729021
HQ728288
HQ728283
EU118668
EU118670
JQ031130
JN710512
JN710513
JN710602
JN710606

JN710607

Miettinen & al. (012
Miettinen & al. 2012
Binder & al. 2013
Miettinen & al. 2012
Binder & al. 2013

TomSsovsky & al. 2010

Miettinen & al. 2012

Sjokvist & al. 2012

Vu & al. 2019

Binder & al. 2013

Miettinen &
Rajchenberg 2012

Miettinen &
Rajchenberg 2012

Miettinen &
Rajchenberg 2012

Present study

Present study

Miettinen & al. 2012

Tomsovsky & al. 2010
Tomsovsky & al. 2010
Tomsovsky & al. 2010
Tomsovsky & al. 2010
Larsson 2007

Sjokvist & al. 2012
Miettinen & al. 2012
Miettinen & al. 2012
Miettinen & al. 2012
Miettinen & al. 2012
Miettinen & al. 2012

Miettinen & al. 2012

652 ... Xu & al.

Systematics of the Polyporales has benefitted from numerous molecular
phylogenetic studies (e.g., Binder & al. 2005, 2013; Larsson 2007; Miettinen
& al. 2012; Dai & al. 2015; Choi & Kim 2017). Steccherinaceae Parmasto, one
of 18 families recognized in Polyporales (Justo & al. 2017), has been included
in several molecular studies (e.g., Binder & al. 2005, 2013; Miettinen & al.
2012; Miettinen & Ryvarden 2016; Justo & al. 2017; Westphalen & al. 2018).
Miettinen & al. (2012) published a multigene, molecular phylogenetic study
of Steccherinum and allied taxa that clearly delineated Steccherinaceae. They
uncovered surprising morphological diversity and plasticity in this family,
requiring revision of generic concepts and 15 new genera to accommodate
existing and new species. Subsequently, Miettinen & Ryvarden (2016)
introduced five new genera, revised one genus, and described two new
species that had been identified earlier by Miettinen & al. (2012). Justo &
al. (2017), who revised family-level classification in Polyporales, confirmed
Steccherinaceae as a distinct lineage in Polyporales that grouped with
Cerrenaceae Miettinen & al. and Panaceae Miettinen & al. Ina morphological
and molecular study of Neotropical taxa of Junghuhnia and Steccherinum,
Westphalen & al. (2018) uncovered a new genus and several new species and
reclassified four taxa.

Cosmopolitan in distribution, Steccherinaceae has a rich diversity because
it is found in boreal, temperate, subtropical, and tropical ecosystems (Nunez
& Ryvarden 2001, Dai 2012, Ryvarden & Melo 2014, Dai & al. 2015, Zhou
& al. 2016). Many new species in Polyporales have been described from
southern, subtropical China (e.g., Li & Cui 2010, Zhao & Wu 2017, Zhao
& Ma 2019). Recently, we collected an undescribed taxon from Yunnan
Province that could not be assigned to any described genus. We present
morphological and molecular phylogenetic evidence that support the
recognition of a new monotypic genus in Steccherinaceae—Rhomboidia,
typified by R. wuliangshanensis.

Materials & methods

The specimens studied are deposited at the herbarium of Southwest Forestry
University, Kunming, China (SWFC). Macromorphological descriptions are based
on field notes. Special colour terms follow Petersen (1996). Micromorphological
data were obtained from the dried specimens and observed under a light microscope
following Dai (2010). The following abbreviations are used: KOH = 5% potassium
hydroxide; CB = cotton blue; CB- = acyanophilous; IKI = Melzer’s reagent; IKI- =
non-amyloid and non-dextrinoid; L = mean spore length (arithmetic average of all
spores); W = mean spore width (arithmetic average of all spores); Q = variation in

Rhomboidia wuliangshanensis gen. & sp. nov. (China) ... 653

the L/W ratios between the specimens studied; (n = a/b) = number of spores (a)
from number of specimens (b).

HiPure Fungal DNA Mini Kit II was used to obtain genomic DNA from dried
specimens, according to the manufacturer’s instructions with some modifications.
A small piece (about 30 mg) of dried fungal material was ground to powder with
liquid nitrogen. The powder was transferred to a 1.5 ml centrifuge tube, suspended
in 0.4 ml of lysis buffer, and incubated in a 65 °C water bath for 60 min. After that,
0.4 ml phenol-chloroform (24:1) was added to each tube and the suspension was
shaken vigorously. After centrifugation at 13,000 rpm for 5 min, 0.3 ml supernatant
was transferred to a new tube and mixed with 0.45 ml binding buffer. The mixture
was then transferred to an adsorbing column (AC) for centrifugation at 13,000 rpm
for 0.5 min. Then, 0.5 ml] inhibitor removal fluid was added in AC for a centrifugation
at 12,000 rpm for 0.5 min. After washing twice with 0.5 ml washing buffer, the AC
was transferred to a clean centrifuge tube, and 100 ml elution buffer was added to
the middle of adsorbed film to elute the genomic DNA. The internal transcribed
spacer region (ITS) was amplified with primer pairs ITS5 and ITS4 (White & al.
1990). The nuclear large subunit region (LSU) was amplified with primer pairs
LROR and LR7 (https://sites.duke.edu/vilgalyslab/rdna_primers_for_fungi/). The
PCR procedure for ITS was: initial denaturation at 95 °C for 3 min, followed by
35 cycles of 94 °C for 40 s, 58 °C for 45 s, and 72 °C for 1 min; and a final extension of
72 °C for 10 min. The PCR procedure for LSU was: initial denaturation at 94 °C for 1
min, followed by 35 cycles of 94 °C for 30 s, 48 °C 1 min, and 72 °C for 1.5 min; and
a final extension of 72 °C for 10 min. The PCR products were purified and directly
sequenced at Kunming Tsingke Biological Technology Limited Company. All newly
generated sequences were deposited at GenBank (TABLE 1).

Sequencher 4.6 was used to edit the DNA sequence. Sequences were aligned
in MAFFT 6 (Katoh & Toh 2008, http://mafft.cbrc.jp/alignment/server/) using the
“G-INS-I” strategy and manually adjusted in BioEdit (Hall 1999). The sequence
alignment was deposited in TreeBase (submission ID 24216). Xanthoporus syringae
(Parmasto) Audet obtained from GenBank was used as an outgroup to root trees
following Miettinen & al. (2012) in the ITS+LSU analyses (Fic. 1).

The ITS+LSU sequences were analyzed phylogenetically using maximum
parsimony, maximum likelihood, and Bayesian inference methods. Maximum
parsimony (MP) analyses followed Zhao & Wu (2017), and tree construction was
performed in PAUP* version 4.0b10 (Swofford 2002). All characters were equally
weighted and gaps were treated as missing data. Trees were inferred using the
heuristic search option with TBR branch swapping and 1000 random sequence
additions. Max-trees was set to 5000, branches of zero length were collapsed and
all parsimonious trees were saved. Clade robustness was assessed using bootstrap
(BP) analysis with 1000 replicates (Felsenstein 1985). Tree statistics tree length
(TL), consistency index (CI), retention index (RI), rescaled consistency index
(RC), and homoplasy index (HI) were calculated for each Maximum Parsimonious
Tree generated. Sequences were analyzed using Maximum Likelihood (ML) with

654 ... Xu & al.

RAxML-HPC2 through the Cipres Science Gateway (www.phylo.org; Miller & al.
2009). Branch support (BS) for ML analysis was determined by 1000 bootstrap
replicates.

MrModeltest 2.3 (Posada & Crandall 1998; Nylander 2004) was used to
determine the best-fit evolution model for data set for Bayesian inference (BI).
Bayesian inference was calculated with MrBayes_3.1.2 using a general time
reversible (GTR+G) model of DNA substitution and a gamma distribution rate
variation across sites (Ronquist & Huelsenbeck 2003). Four Markov chains were run
for 2 runs from random starting trees for 4 million generations (ITS+LSU) in Fic.
1 and trees were sampled every 100 generations. The first 25% of the generations
were discarded as burn-in. A majority rule consensus tree of all remaining trees was
calculated. Branches that received bootstrap support for maximum likelihood (BS)
275%, maximum parsimony (BP) 275%, and Bayesian posterior probabilities (BPP)
20.95 were considered significantly supported.

Molecular phylogenetic results

The ITS+LSU (Fic. 1) dataset comprised sequences from 55 fungal
specimens representing 34 taxa, including the outgroup taxon. The dataset
had an aligned length of 2296 of which 1395 were constant, 201 parsimony-
uninformative, and 700 parsimony-informative. MP analysis yielded two
equally parsimonious trees (TL = 3897, CI = 0.376, HI = 0.624, RI = 0.627, RC
= 0.236). The best-fit model for ITS+LSU alignment estimated and applied
in the BI was GTR+I+G, lIset nst = 6, rates = invgamma; prset statefreqpr =
dirichlet (1,1,1,1). BI resulted in a similar topology with an average standard
deviation of split frequencies equal to 0.006862.

Rhomboidia wuliangshanensis forms a monophyletic lineage with strong
support (BS = 100%, BP = 100%, BPP = 1) and is sister to the Nigroporus-—
Trullella clade (Fic. 1).

Taxonomy

Rhomboidia C.L. Zhao, gen. nov.
MB 833318

Differs from Nigroporus and Trullella by its stipitate to substipitate basidiomata, its
orange-brown to reddish brown surface, and its monomitic hyphal system in both
context and trama.

Fic. 1. Maximum parsimony strict consensus tree illustrating the phylogeny of Rhomboidia
wuliangshanensis and related species in the residual polyporoid clade based on ITS+nLSU
sequences. Branches are labeled with maximum likelihood bootstrap >70%, parsimony bootstrap
proportions >50% and Bayesian posterior probabilities >0.95.

Rhomboidia wuliangshanensis gen. & sp. nov. (China) ... 655

G Cerrenaceae
+ Hyphodermataceae

© Inpicaceae

© Ischnodermataceae

@ Meripilaceae 95/95/1

A Meruliaceae -/-/l

& Panaceae - /81/0.95

84/81/1

100/100/1

100/100/1

Physisporinus vitreus CBS 486.72 @
Irpex lacteus CBS 431.48 @

Irpex lacteus DO 421/951208 @
Ischnoderma benzoinum KHL 12099
Ischnoderma resinosum FD-328 ©

Diplomitoporus flavescens X 84 *

Climacocystis borealis KH 13318 @®

Panus strigellus TNPA 243940 &

Panus conchatus X 1234 &

100/100/1

| Steccherinaceae

@ > Fomitopsidaceae

WME P, Leo} 0/100/1|Pseudolagarobasidium acaciicola CBS 115543 @
ee ae ae Pseudolagarobasidium belizense CFMR DLC 04-31 @
x Polyporaceae Oe Pseudolagarobasidium acaciicola CBS 115544 @©

Hypochnicium lyndoniae NL 041031 +
Hypochnicium bombycinum MA 15305 +
95/63/ -| Spongipellis spumeus BRNM 712630 ©

S782 100/100/1

100/100/1

100/100/1

100/91/1
97/61/1

100/100/1

92/94/1

100/88 1! 1 o9/10071

100/58/0.98

100/100/1

Spongipellis spumeus BRNM 734877 &
Spongipellis spumeus PRM 891931 @&

100/99/1)Physisporinus vitreus KHL 11959 (GB) @

Physisporinus vitreus 3163 @

Physisporinus sanguinolentus BRNM 699576 @
Podoscypha venustula CBS 65684 pea
Abortiporus biennis TFRI 274 Ht

100/63/ - |Loweomyces fractipes X 1253 na
10010011 roweomves fractipes X 1250 [jj
Loweomyces fractipes X 1149 [J
100/100/1) Junghuhnia crustacea X 262
Junghuhnia crustacea X 1127 Jj
Flaviporus brownii X 1216
Flaviporus liebmannii X 666 Ij
Flaviporus liebmannii X 251 fj
100/100/I' Flaviporus liebmannii X 249 fj
100/100/1 p Bigpirapers ailaoshanensis CLZhao 595 [ij
Elaphroporia ailaoshanensis CLZhao 596 |
95/100/1- Trullella duracina X290
Trullella polyporoides X510
Trullella dentipora X 200 B
Nigroporus vinosus BHS2008-100 [ij
Nigroporus vinosus 8182 Q
Nigroporus vinosus X 839 fj

100/100/1; Rhomboidia wuliangshanensis CLZhao 4406 BH
Rhomboidia wuliangshanensis CLZhao 441 Ij
Flabellophora sp.X340
Mycorrhaphium adustum Dai 10173 [i
Mycorrhaphium adustum 8024 [fj

100/99/1 - Junghuhnia micropora Spirin 2652 fj
100/100/1 | l Antrodiella sp. X 418 Pil
100/100/1 Skeletocutis novae-zelandiae Ryvarden 38641 ij
Frantisekia mentschulensis BRNM 710170 i |
00/100/1 Fy antisekia mentschulensis 1377 |
Steccherinum fimbriatum KHL 11905 ia
Antrodiella semisupina FCUG 960 [ij
Antrodiella americana Gothenburg 3161 [fj
Steccherinum ochraceum KHL 11902
100/100/l' steecherinum ochraceum Rybergs.n. [i

Xanthoporus syringae X 339 Oo

an Xanthoporus syringae Cui 2177

Xanthoporus syringae Gothenburg 1488 [J

100/100/1

——i

50

656 ... Xu & al.

TYPE SPECIES: Rhomboidia wuliangshanensis C.L. Zhao

EryMoLoGy: Rhomboidia (Lat.): referring to the rhomboid pileus of the basidiocarp

with the poroid hymenophore.
BASIDIOMATA annual, stipitate. Pileus rhomboid, arising from a multiple
branched stipe. Pores angular, small, dissepiments thin, entire. Hyphal system
monomitic; generative hyphae thick-walled bearing clamp connections,
IKI-, CB-; tissues unchanged in KOH. Cystidia absent, fusoid cystidioles
numerous; hyphal ends numerous. Basidia barrel-shaped to clavate.
Basidiospores broadly ellipsoid, hyaline, thin-walled, smooth, IKI-, CB-.

TYPE OF ROT: white rot.

Rhomboidia wuliangshanensis C.L. Zhao, sp. nov. Fics 2, 3
MB 833320

Differs from Nigroporus vinosus by its stipitate to substipitate basidiomata and
monomitic hyphal structure.

Type: China. Yunnan Province: Puer, Jingdong County, Wuliangshan National Nature
Reserve, on angiosperm trunk, 6 Oct 2017, CLZhao 4406 (Holotype, SWFC 0004406;
GenBank MK860715, MK860710).

EryMo.Locy: The specific epithet wuliangshanensis (Lat.) refers to the type locality,

Wuliangshan.
BASIDIOMATA annual, stipitate to substipitate. Pileus rhomboid, arising from
a multiple branched stipe, edges curling slightly inward, 3.5 cm from the base
to margin, 4 cm wide, up to 3 mm thick; pileus surface radially striate, slightly
brown to orange brown when fresh, drying brown to reddish; the margin
acute, entire. Pore surface white when fresh, cream to buff upon drying. Pores
angular, 7-9 per mm, dissepiments thin, entire. Context corky, white, thin, up to
0.5 mm thick. Tubes concolorous with pore surface, corky, up to 2.5 mm long.

- TYPE OF ROT: white rot.

ADDITIONAL SPECIMEN EXAMINED: CHINA. YUNNAN PROVINCE. Puer:
Jingdong County, Wuliangshan National Nature Reserve, on angiosperm trunk,
6 Oct 2017, CLZhao 4411 (SWFC 004411; GenBank MK860716, MK860711).

Discussion

Rhomboidia is supported as a new genus by phylogenetic analyses and
morphological characters. It is embedded in Steccherinaceae with strong
support. Phylogenetically, Rhomboidia is closely related to Flabellophora
G. Cunn., Nigroporus Murrill, and Trullella Zmitr. based on ITS+LSU nuclear
RNA gene analyses (Fic. 1), which is similar to the previous multigene sequence-
based study (Miettinen & al. 2012). The genera closely related to Rhomboidia

Rhomboidia wuliangshanensis gen. & sp. nov. (China) ... 657

Fic. 2. Rhomboidia wuliangshanensis (holotype, SWFC 0004406). Scale bars = 5 mm.

658 ... Xu & al.

Leciiiian

G (a)

t

a

i

Fic. 3. Rhomboidia wuliangshanensis (drawn from the holotype, SWFC 0004406). A. Basidiospores;

B. Basidia and basidioles;

subiculum. Scale bars: a

C. Cystidioles; D. Hyphal ends; E. Hyphae from trama; F. Hyphae from

= 5 um; b-f = 10 um.

Rhomboidia wuliangshanensis gen. & sp. nov. (China) ... 659

are easily separated morphologically: In Flabellophora basidiomata arise
from a submerged pseudosclerotium and develop unilateral pilei with a
crust and a coriaceous context (Nufez & Ryvarden 2001). Nigroporus differs
from Rhomboidia by developing resupinate to pileate basidiocarps with
vinaceous brown to pink or violet pore surface and a dimitic hyphal system
(Gilbertson & Ryvarden 1987). In Trullella basidiomata are spathulate and
light-coloured, with a monomitic hyphal system in the context but dimitic
in the trama (Miettinen & al. 2012, Zmitrovich 2018).

Rhomboidia resembles other stipitate genera in Polyporales such as
Abortiporus Murrill, Jahnoporus Nuss, and Polyporus P. Micheli ex Adans.
Abortiporus, however, has a duplex structure and thick-walled basidiospores
(Nunez & Ryvarden 2001). Jahnoporus is characterized by large spindle-
shaped basidiospores (Gilbertson & Ryvarden 1987), and Polyporus has a
dimitic hyphal system (Bernicchia & Gorjon 2010).

Polypores are extensively studied and well-known in North America
(Gilbertson & Ryvarden 1987, Zhou & al. 2016) and Eurasia (Nufez &
Ryvarden 2001, Bernicchia & Gorjén 2010, Dai 2012, Ryvarden & Melo
2014, Dai & al. 2015), but Chinese polypore diversity is still being explored,
especially in subtropical and tropical areas. Rhomboidia wuliangshanensis
was collected from Yunnan Province, where many new taxa in Polyporales
and Hymenochaetales have been described (e.g., Li & Cui 2010, He &
Li 2011, Yu & al. 2013, Yang & He 2014, Zhao & Wu 2017, Zhao & Ma
2019). We anticipate that additional, undescribed polypore taxa will be
discovered throughout China after extensive collections are analyzed both
morphologically and molecularly.

Acknowledgments

Special thanks are due to Drs. Karen K. Nakasone (Center for Forest Mycology
Research, Northern Research Station, U.S. Forest Service, USA) and Lu-Sen Bian
(Experiment Center of Forestry in North China, Chinese Academy of Forestry,
P.R. China) who reviewed the manuscript. The research is supported by the
National Natural Science Foundation of China (Project No. 31700023) and Yunnan
Agricultural Foundation Projects (2017FG001-042) and the Yunnan Provincial
Innovation Team on Kapok Fiber Industrial Plantation (2018HC014) and the
Science Foundation of Education Department in Yunnan (2018JS326).

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MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 663-675
https://doi.org/10.5248/134.663

Filsoniana lhasanensis sp. nov.
from Tibet, China

XuE-MEI WEN”?, HURNISA SHAHIDIN'?, ABDULLA ABBAS!

‘College of Life Science and Technology, Xinjiang University,
Urumai, 830046, P. R. China
*Tibet Plateau Institute of Biology, Lhasa, 850001, P. R. China

* CORRESPONDENCE TO: hurnisa_xju@sina.com, zxg_lichen@163.com

ABSTRACT—A new species, Filsoniana lhasanensis, is described from Tibet, China, where it
grows on rocks at altitudes 4200-4432 m. The lichen is characterised by a poorly developed or
absent dark orange areolate thallus, lecanorine to zeorine orange to deep-orange aggregated
apothecia, and richly branched and anastomosed paraphyses. DNA was obtained for three
gene loci—nuclear internal transcribed spacer (ITS), nuclear large subunit (LSU), and
mitochondrial small subunit (SSU). Phylogenetic analyses support the taxon as a new species
of Filsoniana.

Key worps—Himalaya, phylogeny, taxonomy, Teloschistoideae

Introduction

Teloschistaceae is a widespread and well-delimited family of lichenized
fungi, estimated at over 1000 species (Arup & al. 2013). Most species
contain anthraquinone (which confers a yellow to orange color) and
grow under sun-exposed conditions (Gaya & al. 2015). Recent molecular
phylogenetic research supports subdivision of the Teloschistaceae into three
subfamilies: Xanthorioideae, Caloplacoideae, and Teloschistoideae (Arup &
al. 2013; Gaya & al. 2015). Kondratyuk & al. (2013, 2015), who evaluated
Teloschistoideae based on three gene loci (ITS, LSU, SSU), proposed an
additional subfamily Brownlielloideae; however this analysis was based
on a hybrid sequence dataset comprising ITS and LSU data derived from

664 ... Wen, Shahidin, Abbas

the lichen mycobiont and the SSU derived from a fungal contaminant
(Vondrak & al. 2018). Many small monophyletic genera established in
Brownlielloideae were based solely on DNA data without any distinguishing
phenotypic characters, and these genera remain within the Teloschistoideae
as proposed by Arup & al. (2013).

During our study on Chinese Teloschistaceae, we collected some
interesting samples from the suburbs of Lhasa and Shannan in Tibet. The
Tibetan plateau is a unique biogeographical region with varied landscapes,
altitudinal belts, and alpine ecosystems and is considered a world center of
species formation (Zhang & al. 2002). Teloschistaceae in this region have
been inadequately studied, with only 15 species reported (Obermayer 2004;
Wei 1991, 2017; Poelt & Petutschnig 1992; Poelt & Hinteregger 1993; Wei
& Jiang 1986). More species are known from the neighboring Himalayan
regions: India, where >74 species of Teloschistaceae have been recorded
(Joshi & Upreti 2006, 2011; Joshi & al. 2008, 2009, 2014; Kondratyuk & al.
2018) and Nepal, where 47 species have been discovered (Olley & Sharma
2013). According to available literature, there are still large underexplored
areas in the Tibet region, and additional undescribed lichenized fungi
species are expected to be discovered in this poorly studied region.

Most of crustose Teloschistaceae species have few taxonomically
significant morphological characters. Traditional species concepts based in
morphology and chemistry are overly simplified for the crustose species
in Teloschistaceae. Modern classification methods are essential for accurate
taxonomic placement of rare and undescribed species. We combined
sequence analyses of three gene loci—ITS, LSU, SsU—with morphological
and chemical methods to describe a new species, Filsoniana lhasanensis.
This is the first report of the genus Filsoniana in China.

Materials & methods

Morphology & chemistry

Specimens collected from Lhasa and Shannan, Tibet, China, were preserved in
Tibet Plateau Institute of Biology, Lhasa, China (XZ) and Lichen Research Center
in Arid Zone of Northwest China, Xinjiang University, Urumqi, China (XJU-L).
The specimens were examined morphologically using a Shunyu SZM45 dissecting
microscope and Nikon Eclipse E100 compound microscope. Colors follow RAL
color system (https://www.ralcolorchart.com/) Sections for anatomical details and
measurements were made manually and mounted in water. Chemical constituents
were identified by thin-layer chromatography using solvent systems C (Orange
& al. 2010). Thallus morphological structures were photographed with a Nikon

Filsoniana lhasanensis sp. nov. (China) ... 665

TABLE 1. Primers and cycling parameters used for PCR.

Locus PRIMERS PCR SETTINGS

ITS ITS1F (Gardes & Bruns 1993) 94°C-5 min; 6 cycles of: 94°C-45 sec, 57-52°C
1 ITS4 (White & al. 1990) : (decreasing 1°C per cycle) -55 sec, 72°C-1 min; 30
:_ cycles of: 94°C-45 sec, 51°C-55sec, 72°C-1 min; and
| 72°C-7 min

LSU { ALIR (Déring & al. 2000) | 94°C-10 min; 6 cycles of: 94°C-1 min, 57-52°C
| LR6 (Vilgalys & Hester 1990) 1 (decreasing 1°C per cycle) -1 min, 72°C-1.2 min; 30
| cycles of: 94°C-1 min, 51°C-1 min, 72°C-1.2 min;
' and 72°C-2 min

ssu mrSSU1 (Zoller & al. 1999) 94°C-10 min; 6 cycles of: 94°C-45 sec, 57-52°C

mrSSU7 (Zhou & Stanosz 2001) : (decreasing 1°C per cycle) —55 sec, 72°C-1 min; 30
1 cycles of: 94°C-45 sec, 51°C-55sec, 72°C-1 min; and
72°C-7 min

DS-Fi2 Digital Camera using a Nikon MZ25 and Nikon Eclipse Ci stereomicroscope.
All measurements were made using the Nikon Nis elements (D). Morphology and
anatomy were described according to Vondrak & al. (2013).

DNA extraction, PCR amplification, DNA sequencing

Total genomic DNA was extracted using the Fungi Genomic DNA Extraction
Kit, following manufacturer's instructions. Molecular data were generated for three
loci: the nuclear internal transcribed spacer (ITS), nuclear large subunit (LSU), and
mitochondrial small subunit (SSU). Primers and PCR cycling parameters used for
amplification are summarized in TABLE 1.

Sequence analysis

The newly generated sequences were compared to GenBank database sequences
using BLASTN search (http://www.ncbi.nlm.nih.gov/BLAST/). All sequences were
aligned with sequences of selected representatives of Teloschistoideae obtained
from GenBank (TABLE 2) using MAFFT version 7 (Katoh & Standley 2013) with
G-INS-i method.

Phylogenies were generated using Bayesian inference and Maximum Likelihood
(ML) methods. Bayesian inference used the best fitting model as inferred by j Model
Test v2.1.6 (Darriba & al. 2012), implemented in MrBayes v3.2.6 (Ronquist & al.
2012) on the Cipres Scientific gateway (Miller & al. 2010), TrNef+I+G for ITS and
LSU and HKY+I+G for SSU. Two parallel MCMCMC runs were executed, each
using eight chains and 10,000,000 generations, with trees sampled every 1000th
generation.

Maximum likelihood (RAxML) analyses were performed using RAxMLHPC
v.8 on XSEDE (Stamatakis 2014) under the GTRGAMMA model on CIPRES
Science Gateway (Miller & al. 2010). Rapid bootstrap analyses were performed
with 1000 bootstrap replicates. The phylogenetic tree was drawn using FigTree ver.
1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

666 ... Wen, Shahidin, Abbas

TABLE 2. Sequences used in the phylogenetic analyses.
(Sequences generated for this study are in bold.)

SPECIES VOUCHER ITS LSU SSU
Brownliella aequata | SK838 [4] | KF264627 | KF264662 { KF264688
! SK831 [4] ! KF264626 ! KF264661 ! KF264687
B. kobeana ! 130318 [5] ! KT456214 ! KT456229 ! KT456244
! 120032 [5] ! KT456212 ! KT456227 ! KT456242
B. montisfracti ! SK231 [4] ! KF264625 ! KF264660 ! KF264686
! SK230 [4] ! KF264624 ! KF264659 ! _
Browniliella sp. SK2013a [4] KF264628 KF264663 ! —
Caloplaca sp. ! Casan 242 [3] ! KT291463 ! KT291558 ! KT291508
! Gaya 110 [3] ! KT291456 ! KT291552 ! KT291499
Elixjohnia bermaguiana ! Kondratyuk 20487 [1] ! KC179299 ! KC179245 ! KC179584
E. gallowayi ! Karnefelt 997504 [1] ! KC179301 ! KC179247 ! KC179586
E. jackelixii ! U1402 [1] ! KC179303 ! KC179248 ! KC179587
! SK910 [4] ! KF264655 ! KF264683 ! KF264707
Filsoniana australiensis ! SK851 [4] ! KF264631 ! KF264665 ! KF264691
! SK850 [4] ! KF264632 ! KF264666 ! KF264692
EF. lhasanensis 224, Wen 20110910-05 MK153161 MK439835 MK439831
! Z19, Wen 20110901-29 ! MK153160 ! MK439834 ! MK439830
E rexfilsonii ! SK859 [4] ! KF264638 ! KF264671 ! —
! SK861 [4] ! KF264636 ! KF264670 ! =
Follmannia orthoclada ! Frodén 1772 [1] ! KC179291 ! KC179191 ! _
Haloplaca sp. ! 6-UA2013 [1] ! KC179295 ! KC179203 ! KC179537
Harusavskia elenkinianoides ! SK997 [6] ! KY614404 ! KY614452 ! KY614485
! SK269 [6] ! KY614405 ! KY614453 ! KY614486
Josefpoeltia parva ! Frédén 1671 [1] ! KC179296 ! KC179204 ! KC179539
J. sorediosa ! Frodén 1593 [1] ! KC179297 ! KC179205 ! KC179540
! SK992 [4] ! KF264646 ! KF264674 ! KF264697
! SK991 [4] ! KF264645 ! KF264673 ! KF264696
Kaernefia albocrenulata ! SK245 [4] ! KF264647 ! KF264675 ! KF264698
! SK246 [4] ! KF264648 ! KF264676 ! KF264699
K. gilfillaniorum ! SK999 [4] ! KF264650 ! KF264678 ! KF264701
! SK253 [4] ! KF264649 ! KF264677 ! KF264700
K. kaernefeltii ! SK919 [4] ! KF264651 ! KF264679 ! KF264702
! SK921 [4] ! KF264652 ! KF264680 ! KF264703
Nevilleiella lateritia | SK 261 [6] 1 KY614427 | KY614464 | KY614502

Filsoniana lhasanensis sp. nov. (China) ... 667

SPECIES VOUCHER ITS LSU SSU

| SK 878 [6] | KY614426 | KY614463 | KY614501
N. marchantii ! SK D18 [6] ! KY614425 ! KY614462 ! KY614500
Scutaria andina ! PF209 [1] ! KC179298 ! KC179242 ! KC179581
Sirenophila cliffwetmorei ! SK A93 [6] ! KY614438 ! KY614471 ! KY614513
S. eos ! U1408 [1] ! KC179300 ! KC179246 ! KC179585
S. gintarasii ! SK D17 [6] ! KY614437 ! KY614470 ! KY614512
S. maccarthyi ! Karnefelt 977801 [1] ! KC179304 ! KC179249 ! KC179588
Solitaria chrysophthalma ! Cchr157 [3] ! KT291446 ! KT291537 ! KT291484
Stellarangia elegantissima ! PF 81 [1] ! KC179310 ! KC179254 ! KC179593

! Cele 75 [3] ! KT291454 ! KT291541 ! KT291488
Teloschistes exilis ! Gaya 12 [2] ! JQ301630 ! JQ301577 ! JQ301519
T. flavicans ! AFTOL-ID 315 [2] ! JQ301578 ! JQ301631 ! JQ301520

! Tflav103 [3] ! KT291472 ! KT291565 ! KT291523
T. hosseusianus Gaya 50 [2] KC179318 ! JQ301579 ! JQ301521
T. hypoglaucus ! PF68 [1] ! KC179319 ! KC179256 ! KC179595
T. sieberianus ! Tsie423 [3] ! EU639655 ! pez ! KT291525
Teloschistopsis bonae-spei ! PF82 [1] ! KC179322 ! KC179257 ! KC179596
Ts. eudoxa ! PF72 [1] ! KC179324 ! KC179258 ! KC179597
Thelliana pseudokiamae SK925 [4] KF264633 KF264667 —

! SK926 [4] ! KF264634 ! KF264668 ! =

! SK 925 [5] ! KT456225 ! KT456240 ! _—

! SK 926 [5] ! KT456226 ! KT456241 ! _—
Villophora isidioclada ! USE563 [1] ! KC179325 ! KC179266 ! KC179606
Villophora sp. 50 ! U1342 [1] ! KC179330 ! KC179268 ! —
Villophora sp. 40 ! USE574 [1] ! KC179328 ! KC179267 ! KC179607
Wetmoreana decipioides Cadec188 [3] KT291453 KT291540 KT291487

! SK689 [4] ! KF264644 ! — ! KF264695
W. texana ! SK536 [4] ! KF264658 ! = ! KF264711

! USE527 [1] ! KC179337 ! KC179273 ! KC179612
W. sp. 52 ! Frédén 1645 [1] ! KC179334 ! KC179270 ! KC179609
W. sp. 54 ! Frédén 1521 [1] ! KC179336 ! KC179272 ! KC179611
Xanthomendoza fallax ! Gaya 33 [2] ! JQ301687 ! JQ301580 ! JQ301633
Xa. ulophyllodes ! T11, Shahidin 145507 ! MK414784 ! MK439836 ! MK439832

! T22, Shahidin 145449 ! MK414785 ! MK439837 ! MK439833
Xanthoria sp. ! Gaya 31 [2] ! JQ301692 ! JQ301591 ! JQ301532

Sources: [1] Arup & al. (2013); [2] Gaya & al. (2012); [3] Gaya & al. (2015);
[4] Kondratyuk & al. (2013); [5] Kondratyuk & al. (2015); [6] Kondratyuk & al. (2017).

668 ... Wen, Shahidin, Abbas

Xanthomendoza ulophyllodes

Xanthomendoza ulophyllodes
Xanthomendoza fallax

1/100) Villophora isidioclada
1/100 illophora sp. 50
Villophora sp. 40
Josefpoeltia parva
q- Josefpoeltia sorediosa
Josefpoeltia sorediosa
1/80 0.99/99L Josefpoeltia sorediosa
1/100 Teloschistes exilis
1/100 , Teloschistes flavicans
1/94 Teloschistes flavicans
Teloschistes sieberianus
oor- Teloschistes hosseusianus
1/100 5 Teloschistes hypoglaucus
Teloschistes chrysophthalmus

1/100 1/100

Caloplaca sp.
+ ~ 219 Filsoniana lhasanenses
© - 224 Filsoniana Ilhasanenses
SK861 Filsoniana rexfilsonii
-/88} SK925 Thelliana pseudokiamae
SK925 Thelliana pseudokiamae
SK926 Thelliana pseudokiamae
SK926 Thelliana pseudokiamae
SK859 Filsoniana rexfilsonii
0.97/86 Filsoniana australiensis
1/100 SK850 Filsoniana australiensis
iy Nevilleiella marchantii
1/9 Nevilleiella lateritia
1/95) 1/99 Nevilleiella lateritia
1/100 Brownliella sp.
1/100 Brownliella aequata
Brownliella aequata
1/100, 8rownliella kobeana
Brownliella kobeana
1/100 Brownliella montisfracti
1/73 Brownliella montisfracti
1/100; Keernefia gilfillaniorum
Kaernefia gilfillaniorum

1/100
°
6

1/93

thi

1/94

001

1/100 », Kaernefia kaernefeltii
SL Kaernefia kaernefeltii
O— Kaernefia albocrenulata
99 1/99 Kaernefia albocrenulata
/ Haloplaca sp.
Elixjohnia bermaguiana
1 1/100 as ; R
1/94 1/100 Elixjohnia gallowayi
1/100 Elixjohnia jackelixii
1/90 AGk Bie a
1/1004 Elixjohnia jackelixii
1/99 Sirenophila eos
1/95 Sirenophila maccarthyi
Sirenophila gintarasii
-/95 1/100 . labbod
enophila cliffwetmor
1/100 Teloschistopsis bonae-spei
Teloschistopsis eudoxa
1/100 Stellarangia elegantissima
1/70 Stellarangia elegantissima
1/100 Wetmoreana decipioides
Wetmoreana decipioides
1/100 1/100 Wetmoreana texana
1/100} Wetmoreana texana
1/95 Wetmoreana sp. 54
-94/90]
0.949 Wetmoreana sp. 52
1/85) 0.95/9 Caloplaca sp.
Solitaria chrysophthaima
1/100 Xanthoria sp.

Scutaria andina
1/100 , Harusavskia elenkinianoides
1/99 Harusavskia elenkinianoides
Follmannia orthoclada

0.95/

ae O09

Fic. 1. The ML tree based on a concatenated 3-loci data matrix. The numbers above each node
represent posterior probability (PP) and bootstrap support (BS) values. Only PP values >0.90
and BS values >70% are shown. The species described in this paper is shown in blue font.

Results & discussion

New ITS, LSU, and SSU sequences were generated in this study. A
total of 70 ITS sequences, 67 LSU sequences, and 58 SSU sequences were
included in the analyses (Fic. 1). Trees of similar topologies were also
generated using the maximum likelihood method and Bayesian approach,

Filsoniana lhasanensis sp. nov. (China) ... 669

with Xanthomendoza ulophyllodes and X. fallax as outgroup. Our combined
analyses grouped our new species with Filsoniana rexfilsonii and Thelliana
pseudokiamae in a monophyletic clade with high support value (BS = 93,
PP = 1). The new species is closely related to Thelliana pseudokiamae
(S.Y. Kondr. & Karnefelt) S.-Y. Kondr.& al. and E rexfilsonii (S.Y. Kondr.
& Karnefelt) S.Y. Kondr. & al.. Almost all other members of Filsoniana
have well-developed thalli and occur in Australia. The sequences listed
by Kondratyuk & al. (2013) under the name “Squamulea kiamae” were
misdetermined; they were derived from specimen Karnefelt 994101 [LD
1101337], which was resequenced by Kondratyuk &al. (2015) and designated
as the holotype of a new genus and species, Thelliana pseudokiamae S.Y.
Kondr. & al. Therefore all four of the “Squamulea kiamae” sequences
represent Th. pseudokiamae, and authentic sequences of S. kiamae are not
included in our analyses. Filsoniana kiamae and Th. pseudokiamae differ in
the sorelia and lobe size (Kondratyuk & al. 2007, 2015). Our results conflict
with Kondratyuk & al. (2015), whose ITS-LSU-SSU combined analyses
placed Filsoniana in Brownlielloideae; our analyses place most members of
Brownlielloideae in the Teloschistoideae.

Taxonomy

Filsoniana lhasanensis X.M. Wen, Shahidin & A. Abbas sp. nov. Fic. 2
FN 570592

Differs from Filsoniana kiamae and E rexfilsonii by its reduced thallus, its aggregated
adnate apothecia, and its Asian distribution.

Type: China. Tibet, Lhasa City, Maizhuokunggar county, Riduo Village, 29°42’35”N
92°05’10’E, alt. 4200 m, 1 September 2011, X. M. Wen 20110901-29 (Holotype, XZ;
isotype, XJU-L; GenBank MK153160, MK439834, MK43983).

EryMo.ocy: The epithet /hasanensis refers to the region in which the type specimen

was collected.
THALLUS crustose, areolate, poorly developed or absent, usually confined
to the apothecia, 0.1-0.2 mm diam., orange to deep orange [RAL 2011].
CorTEX paraplectenchymatous, 12.9-67.1 um high, composed of thin-
walled cells, (2.38-)4.8-7.4(-10.5) um diam. ALGAL LAYER + continuous,
49.5-97.9 um thick. MEDULLA loosely interwoven by thin-walled hyphae
under the apothecia, 59.8-86.1 um high.

APOTHECIA lecanorine to zeorine, numerous, adnate, always

aggregated (2-7) or crowded together, disc margins present, smooth,
entire, concolorous with thallus, sun yellow [RAL 1037] to deep orange

670 ... Wen, Shahidin, Abbas

[RAL 2011], disc round to irregular, yellow-orange [RAL 2000] to signal
orange [RAL 2010], flat to concave, 0.3-0.7 mm diam. EXxcIPLE 9.6-14.8
um thick, consisting of 2-4 layers of cells, upper part of cells spherical,
3.2-6.0 um diam, cells of lower part elongated, 3.5-10.6 x 1.4-3.6 um.
HypoTuHEcium thin, 22.3-34.7 um high, composed of several lines of
paraplectenchymatous cells, 2.9-5.2 x 2.3-4.6 um. HYMENIUM 52.2-89.5
um high, colorless. PARAPHYSES anastomosed, generally dichotomously
branched or trigeminally branched at tips, the 3-4 terminal cells gradually
enlarging towards tips, the widened cells nonuniform in size, the second
cells 3.5-6.7 um diam., and the third ones 3.2-5.3 um diam.

Ascr_ teloschistes-type, 8-spored. Ascospores colorless, ellipsoid,
(11.2-)11.7-17.5(-4.7) x 5.8-8.4(-9.3) um, septum 3.3-6.5 um diam.

SPOT TESTS—K+ red.
CHEMISTRY—Parietin, emodin, and two unknown anthraquinones.

EcoLtocy—Filsoniana lhasanensis grows on rocks in open areas
and associates with species of Aspicilia, Lecanora, Rusavskia, Physcia,
Rinodina, Rhizoplaca, and Acarospora. The new lichen is distributed
in arid or semiarid areas of Lhasa city and Shannan city at elevations of
4200-4432 m.

ADDITIONAL SPECIMEN EXAMINED: CHINA. TIBET, Shannan City, Nagarze county,

Yamzho Yumco, 29°09’34”N 90°30'19’E, alt. 4432 m, 10 September 2011, X.M. Wen

20110910-05 (XZ; GenBank MK153161, MK439835, MK439831).
REMARKS: Filsoniana lhasanensis is characterized by a reduced, dark orange
to brownish orange thallus and aggregated apothecia. It is morphologically
similar to Xanthocarpia ferrarii (Bagl.) Frodén & al., which differs by its
narrower septum, larger ascospores, well developed true exciple, zeorine
to biatorine apothecia, and classification in Xanthorioideae (Arup & al.
2013).

Filsoniana australiensis, F. rexfilsonii, and F. lhasanensis have similar
paraplectenchymatous cortex and broad paraphyses (Kondratyuk & al.
2007). However, F. australiensis and E rexfilsonii have well-developed thalli
(E australiensis has a placodioid thallus, and FE rexfilsonii has a squamulose
thallus) with scattered or rare apothecia, whereas F [hasanensis has a poorly
developed thallus consisting of only a few scattered areoles.

A summary of the characteristics of FE [hasanensis and morphologically
similar species is provided in TABLE 3.

Filsoniana lhasanensis sp. nov. (China) ... 671

Fic. 2. Filsoniana lhasanensis (XZ (Wen 20110901-29)): A. Thallus and apothecia; B. Cortex
of thallus; C. Cross section of the apothecium; D. Hypothecium; E. Paraphyses branched and
enlarged at the tips; F. Paraphyses branched and anastomosed; G. Ascus containing eight
ascospores. Scale bars: A = 1 mm; B-F = 10 um; G=5 um.

672 ... Wen, Shahidin, Abbas

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‘pedoyaaap Aj100d Jo Juasqy ‘moTad YsTUMoIg ‘SUIBsIe|y Ajr00d 30 yuasqy ‘padopaaap [aM ‘prorpooryd ‘padojaaap [aM snyeqL,
I I I I I
sisuauvsvY] “I aviuviyopnasd ‘T, MADLsaf "X 1MOS]YXaL + SISUAITDAISND + WILOVAVHD

vid VIOYJUVY pure “‘vubIYaY], “vU_IUOS]1T JO Sotdods sIeTIUMTs ATTeOISojoydiour YIM sisuauvsvYy] DULIUOS]1.J JO UOSTIedWIO’) ‘¢ ATAVI,

Filsoniana lhasanensis sp. nov. (China) ... 673

Acknowledgments

This study was supported by the National Natural Science Foundation of China
(31093440, 31260008) and Natural Science Foundation of Xizang Autonomous
Region, China (2016ZR-QY-05). The authors are grateful to Dr. Jan Vondrak
(Institute of Botany, Academy of Sciences of the Czech Republic, Prihonice) and
Prof. Xinli Wei (State Key Laboratory of Mycology, Institute of Microbiology,
Chinese Academy of Sciences, Beijing) for peer review.

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MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019— Volume 134, pp. 677-679
https://doi.org/10.5248/134.677

Sarcopodium flocculentum,
the correct name for S. macalpinei

SHAUN R. PENNYCOOK" & PAUL M. KIRK?

'Manaaki Whenua - Landcare Research

Private Bag 92170, Auckland 1072, New Zealand
? Royal Botanic Gardens

Kew, Surrey, TW9 3DS, UK

* CORRESPONDENCE TO: PennycookS@LandcareResearch.co.nz

ABSTRACT— The nomenclatural background of the species treated as Sarcopodium macalpinei
is briefly outlined, and the requirement to supersede that name with a combination based on
Nectriella flocculenta [= Lanatonectria flocculenta] is explained.

Key worps—Actinostilbe flocculenta, Actinostilbe macalpinei, Kutilakesopsis macalpinei,
ICN, code-compliance

In a morphological study, Sutton (1981) synonymised Actinostilbe Petch
and Kutilakesopsis Agnihothr. & G.C.S. Barua with the earlier genus Sarco-
podium Ehrenb. and published a new combination Sarcopodium macalpinei
for the type of Kutilakesopsis.

Rossman & al. (1999) proposed Lanatonectria Samuels & Rossman,
typified by a sexual morph for which they published the new combination
L. flocculenta, additionally proposing a new combination Actinostilbe
macalpinei for the conspecific asexual morph.

In a multi-gene analysis, Lombard & al. (2015) included sequences from
the type species of Sarcopodium (S. circinatum), Actinostilbe (A. vanillae =
S. vanillae), and Kutilakesopsis (K. macalpinei = S. macalpinei), demonstrating
that they were all included within a monophyletic Sarcopodium clade,
thereby confirming the synonymy of these three genera. They also included

678 ... Pennycook & Kirk

Lanatonectria as a synonym based on the established synonymy of its
type (L. flocculenta) with Sarcopodium macalpinei; consequently, they
recombined four of the five Lanatonectria species in Sarcopodium but did
not recombine L. flocculenta (presumably because of its existing heterotypic
Sarcopodium synonym).

Rossman & al. (2016) recommended Sarcopodium as the accepted
generic name in preference to Lanatonectria and Actinostilbe, a decision
based on priority, widespread use, and the greater number of existing
names; but they continued to cite L. flocculenta as S. macalpinei.

However, Sarcopodium macalpinei is not the correct name for this
species, since it fails to use the epithet of the earliest available legitimate
name, as required by ICN (Shenzhen) Art. 11.4 (Turland & al. 2018). The
correct name has been proposed by Pennycook & Kirk (2019):

Sarcopodium flocculentum (Henn. & E. Nyman) Pennycook & P.M. Kirk,
Index Fungorum no. 418: 1. 2019
= Nectriella flocculenta Henn. & E. Nyman, Monsunia 1: 160. 1899 [“1900”]
= Nectria flocculenta (Henn. & E. Nyman) Hohn., Sitzungsber. Kaiserl.
Akad. Wiss., Wien, Math.-Naturwiss. Cl., Abt. 1, 121: 360. 1912
= Lanatonectria flocculenta (Henn. & E. Nyman) Samuels
& Rossman, Stud. Mycol. 42: 138. 1999
= Actinostilbe flocculenta (Henn. & E. Nyman) Rossman,
Samuels & Seifert, IMA Fungus 4(1): 46. 2013
= Kutilakesopsis macalpinei Agnihothr. & G.C.S. Barua, J.
Indian Bot. Soc. 36: 309. 1957 [as ‘macalpineae’ |
= Sarcopodium macalpinei (Agnihothr. & G.C.S. Barua) B.
Sutton, Trans. Brit. Mycol. Soc. 76: 99. 1981
= Actinostilbe macalpinei (Agnihothr. & G.C.S. Barua) Seifert
& Samuels, Stud. Mycol. 42: 138. 1999

Acknowledgments
We wish to thank Tom May (Royal Botanic Gardens, Melbourne) and Scott
Redhead (Agriculture and Agri-Food Canada, Ottawa) for refereeing the manuscript.

Literature cited

Lombard L, van der Merwe NA, Groenewald JZ, Crous PW. 2015. Generic concepts in
Nectriaceae. Studies in Mycology 80: 189-245. https://doi.org/10.1016/j.simyco.2014.12.002

Pennycook SR, Kirk PM. 2019. Nomenclatural novelties: S$. Pennycook & P.M. Kirk. Index
Fungorum no. 418. 1 p.
http://www.indexfungorum.org/Publications/Index%20Fungorum%20n0.418.pdf

Rossman AY, Samuels GJ, Rogerson CT, Lowen R. 1999. Genera of Bionectriaceae, Hypocreaceae
and Nectriaceae (Hypocreales, Ascomycetes). Studies in Mycology 42. 248 p.

Sarcopodium flocculentum (= S. macalpinei) explained ... 679

Rossman AY, Allen WC, Braun U, Castlebury LA, Chaverri P, Crous PW, Hawksworth
DL & al. 2016. Overlooked competing asexual and sexually typified generic names of
Ascomycota with recommendations for their use or protection. IMA Fungus 7: 289-308.
https://doi.org/10.5598/imafungus.2016.07.02.09

Sutton BC. 1981. Sarcopodium and its synonyms. Transactions of the British Mycological
Society 76: 97-102. https://doi.org/10.1016/s0007-1536(81)80012-5

Turland NJ, Wiersema JH, Barrie FR, Greuter W, Hawksworth DL, Herendeen PS & al.
2018. International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code)
adopted by the Nineteenth International Botanical Congress, Shenzhen, China, July 2017.
Regnum Vegetabile 159. https://doi.org/10.12705/Code.2018

MYCOTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 681-705
https://doi.org/10.5248/134.681

Pteridicolous ascomycetes from a cloud forest
in eastern Mexico

ROSARIO MEDEL-ORTIZ?, YAJAIRA BAEZA?,
& FRANCISCO G. LOREA-HERNANDEZ?

‘Centro de Investigacion en Micologia Aplicada, Universidad Veracruzana,
Médicos No. 5, Col. Unidad del Bosque, 91010, Xalapa, Veracruz, México.
*Facultad de Agronomia, Universidad Veracruzana,
Zona Universitaria, 91000, Xalapa, Veracruz, México
*Instituto de Ecologia, A.C. Red de Sistemdtica y Biodiversidad,
Carretera antigua a Coatepec 351, 91070, Xalapa, Veracruz, México.

“CORRESPONDENCE TO: romedel@uv.mx

ABSTRACT— Twenty-one ascomycetous taxa collected on ferns in a cloud forest of Veracruz,
Mexico are reported and described. Fourteen represent new records for the Mexican
mycobiota: Bisporella pteridicola, Crocicreas quinqueseptatum, Dasyscyphella dryina, Durella
macrospora, Hamatocanthoscypha helicotricha, Hyaloscypha fuckelii, Lachnum brevipilosum,
L. nudipes, Mollisia ventosa, Orbilia auricolor, Protocreopsis pertusa, Seimatosporium tostum,
Stictis carnea, and S. radiata. Two collections—Cyathicula and cf. Arachnopeziza—that could
not be fully identified may represent new species. The remaining five taxa have previous
Mexican records but are reported here from new fern hosts. This study increases to 25 the
number of ascomycete species known to occur on ferns in the cloud forests of Mexico.

Key worps—Cyatheaceae, Helotiales, Hypocreales, Ostropales, Xylariales

Introduction

The Ascomycota, with around 90,000 species (Cannon & al. 2018), is the
most diverse group in kingdom Fungi. Its diversity significantly complicates
gaining knowledge of individual species, especially those with minute
fruitbodies restricted to specialized substrates. Such is the case of fungi
growing on ferns. Most fungi reported on ferns are saprobes, a group that

682 ... Medel-Ortiz, Baeza, Lorea-Hernandez

receives less attention than parasites (Mehltreter et al. 2010). Currently
69 ascomycete species have been recorded in cloud forest of Veracruz
(Medel 2013), small in comparison to the over 500 fern species known
to inhabit cloud forests (Tejero-Diez & al. 2011). Although the first fern-
related fungal study was devoted to rust fungi (Stevenson 1945), subsequent
studies have been more focused on ascomycetes and their anamorphs (e.g.,
Mycosphaerellaceae and Periconiella; Braun 2004, Kirschner & Liu 2014).
Other studies have centered on endophytic ascomycetes and fern-associated
saprobes (Cannon 1997, Castaheda-Ruiz & Heredia 2000, Del Olmo &
Arnolds 2014, Dingley 1972, Medel & Lorea-Hernandez 2008, Samuels &
Rogerson 1990).

For Mexico there are relatively few studies on the diversity of fern-
inhabiting fungi (Castafieda-Ruiz & Heredia 2000, Haines 1980, Medel &
Lorea-Hernandez 2008, Medel & al. 2010, Samuels & Rogerson 1990). So
far, the seven ascomycete species recorded growing on ferns represent only
three genera: Crocicreas Fr. (1), Dimeriella Speg. (1), and Lachnum Retz. (5).
The few Ascomycota recorded from these forests contrasting with the great
diversity of fern species suggests a high diversity yet to be explored. The
present study was conducted in order to provide more information about
the diversity of fern-inhabiting fungi in Mexican cloud forests.

Materials & methods

Sampling sites in central Veracruz state (eastern Mexico) were selected based
on the high fern diversity within an area. Three sampling sites were chosen: El
Riachuelo, Los Encinales, and Rio Xocoyolapan. Field work was conducted during
August-November 2011. Fungi were collected by opportunistic sampling of
different fern species, with fronds and rachises presenting ascomata or stromata
selected and placed in paper bags (Mueller & al. 2004).

Fruiting bodies were identified and processed following standard mycological
techniques for Ascomycota (Dennis 1978). Specimens were hand _ sectioned,
pretreated with 3% aqueous KOH, and stained with phloxine and Melzer’s reagent;
ascal tip bluing reactions are designated by J- (negative, no blue present) and J+
(positive, blue present). Tissues were examined microscopically using a Zeiss Primo
Star microscope. Averages, ranges, and Q-calculations (Largent & al. 1977) are
based on 30 measurements. Colours are coded according to Kornerup & Wanscher
(1978). Ascomata were photographed digitally with a Sony Cyber-Shot camera
mounted on a Zeiss Stemi DV4 stereomicroscope. The specimens were identified
using specialized literature (Breitenbach & Kranzlin 1984, Carpenter 1981, Dennis
1949, 1956, 1960, 1978). Fungal nomenclature follows Index Fungorum (2019),
and host plants were identified following Mickel & Smith (2004). All specimens are

Pteridicolous ascomycetes (Mexico) ... 683

deposited in the mycological collection of the Herbarium, Instituto de Ecologia A.C.
(XAL). Specimens discovered in XAL and also examined are noted as “additional
specimens examined” in species descriptions where pertinent.

Taxonomy

cf. Arachnopeziza FIGS 1-3
APOTHECIA sessile, cup-shaped, reddish-golden (6C7) to Pompeian
yellow (5C6), <1 mm diam., margin hairy (sparsely pilose), hairs white
(1A1), readily dropping from the margin to give a smooth appearance.
SUBICULUM white to pale yellowish white (1A1, 2A1), not scattered on the
substrate, reticulate or net-like, with hyphae 2-3 um diam., septate, hyaline,
thick-walled (<1 um). Ascr cylindrical, 70-90(-100) x 7-9(-10) um, thin-
walled, hyaline, 1-2-seriate, pore J+. Ascospores cylindrical-ellipsoid,
(13-)14-15 x 5 um (Q = 2.8), smooth, hyaline, 0-1-septate, biguttulate,
with sheath and hyaline appendages observed in Cresyl blue. PARAPHYSES
filiform, 1-2 um diam., hyaline, septate toward the base. EcraL ExcrpuLUM
a textura globulosa to textura angularis, cells 12—25(—40) um diam.
HaBItTat—Gregarious, on rachises of Alsophila firma. Known only from

these collections.

SPECIMENS EXAMINED—MEXICO. VERACRUz: San Andrés Tlalnelhuayocan County,

Rio Xocoyolapan, 19°30.89’N 97°00.39’W, 1590 m, 13 October 2011, Cordova 78.

ADDITIONAL SPECIMENS EXAMINED: MEXICO. Veracruz: San Andrés

Tlalnelhuayocan County, Rancho Agitita Fria, 6 April 2007, Medel 1362; 1363-A.
Arachnopeziza Fuckel is characterized by small apothecia growing on a
densely reticulate, net-like subiculum covered by hairs, and ellipsoid-fusoid
to filiform, 1-7-septate ascospores (Korf 1951). The genus has a worldwide
temperate distribution but is uncommon in the tropics (Gamundi &
Giaiotti 1994). Although the studied specimens suggest an affinity with
Arachnopezizaceae, they do not match any known genus or species covered
in the references consulted (Dennis 1949, Huhtinen 1987, Korf 1951). The
0-1-septate spores covered by a hyaline sheath with a small appendage
resemble those of Arachnopeziza aurelia (Pers.) Fuckel, which differs in its
long hairs, slightly larger spores (17-22 x 3.5-5.5 um, O0-3-septate; Korf
1951). The presence on the apothecial margin of very thick, scarce, and
septate hairs that drop off, leaving a smooth margin (as in our specimen;
Fic. 1) is a feature unknown in Arachnopeziza. The subiculum is restricted
to the apothecial base and not distributed throughout the substrate, unusual
in Arachnopeziza (Seaver 1938), but not uncommon in Parachnopeziza Kort.

684 ... Medel-Ortiz, Baeza, Lorea-Hernandez

In Parachnopeziza, however, apothecia grow directly from a small stipe
immersed in the substrate (Korf 1978) growing directly from a subiculum, as
occurs in Arachnopeziza, and certainly in our specimen.

Bisporella pteridicola F. Ren & W.Y. Zhuang,
Mycosystema 36 (4): 408 (2017). FIGS 4, 5

APOTHECIA superficial, cup-shaped to discoid. Disc concave, yellow
(4A6), pastel yellow (2A4), or chicken yellow (2A6), translucent, waxy in
appearance, 0.2-0.4 mm diam. Stipe short or absent, concolorous with the
disc. Asci claviform (32—)35-40(-45) x 5-6 um, 8-spored, biseriate, apical
pore J-, arising from croziers. AScosPoREs fusiform (5 —)8-9(-10) x 2-3
um (Q = 4.3), hyaline, with two oil drops and one septum visible in KOH.
PARAPHYSES filiform, slightly enlarged at the apex, 1.5-2 um diam. ECTAL
EXCIPULUM a textura angularis, gelatinized, 15-40 um thick, cells 3-6 um
diam., protruding from outer surface <3 um. MEDULLARY EXCIPULUM a
textura intricata, thin, hyaline, not gelatinized, cells 2-3 um diam.

HasitatT—On rachises of unidentified fern as well as on fern rachises
and leaf blades of Alsophila firma, Cyathea bicrenata, Sphaeropteris horrida,
Pecluma alfredii, and Thelypteris sp.

SPECIMENS EXAMINED—MEXICO. VeRAcRUz: San Andrés Tlalnelhuayocan
County, Rio Xocoyolapan, 19°30.89’N 97°00.39’W, 1590 m, 13 October 2011,
Cordova 80c; 4 November 2011, Castillo del Moral 63, 66; 8 December 2011, Medel
& Lorea 2203. El Riachuelo; 19°30.99’N 97°00.39’W, 1626 m, 18 August 2011, Baeza
47a, Cordova 67, 71; 11 November 2011, Medel 2192. Los Encinales, 19°31.10’N
97°00.32’W, 1630 m, 10 August 2011, Baeza 36; 18 August 2011, Baeza 49,55, 57;
Medel & Lorea 2158, Cérdova 73, 74.

Bisporella Sacc. is a genus easily recognized by its small sessile bright yellow
apothecia growing on woody substrata (Carpenter & Dumont 1978a,
Dumont 1981). Bisporella pteridicola was described growing on ferns in Asia
(Zhuang & al. 2017). Ours is the first report of the species from Mexico.
Our collections agree with the description in Zhuang & al. (2017) in color,
size, and shape of the apothecia, ascospore measurements, and ecology
(growth on ferns).

Coenogonium botryosum C. Knight,
Syn. Queensl. Fl. 1(Suppl.): 74 (1886). Fics 6, 7

APOTHECIA superficial, discoid, sessile, flattened, 0.5-1 mm diam., pale
yellow (3A3), waxy in appearance, margin differentiated from the disc,
smooth, yellowish white (3A2), paler than the disc. THaLtus filamentous
or felt-like and pilose, with short, upright algal threads resembling hairs,

Pteridicolous ascomycetes (Mexico) ... 685

greyish yellow to linden green (2B5-2C5). Hymenium J+. Harrs small
and thin, linden green (2C5) to pale yellow (1A3-2A3), filiform, septate.
Ascli cylindric-clavate (48—)50-55(-60) x 4-5 um, thin walled, uniseriate.
Ascosporss ellipsoid (7-)8-9(-12) x 2 um (Q = 4.2), hyaline, smooth,
0-1-septate, with 4 small guttules. ParApHysEs cylindrical, apices 4-5 um
diam., bifurcate, hyaline, septate. ECTAL EXCIPULUM a textura globulosa,
with hyaline cells.
Hasitat—Gregarious, on fronds of Adiantum andicola and

Grammiitis sp.

SPECIMENS EXAMINED—MEXICO. VERACRUz: San Andrés Tlalnelhuayocan County,

El Riachuelo, 19°30.99’N 97°00.39’W, 1626 m, 8 December 2011, Baeza 70, Medel &

Lorea 2211, 2215.
Coenogonium Ehrenb. is a tropical and subtropical ascolichen genus found
on all continents (GBIF 2019, Rivas-Plata & al. 2006, Uyenco 1963). The
filamentous green-pale yellowish thallus attached to the substrate by small
filaments as well as the threadlike algae, absence of septa, uniseriate asci, and
paraphyses with capitate subglobose apices are characteristic of C. botryosum
(Rivas-Plata & al. 2006). Two similar species are C. moniliforme Tuck., which
has moniliform algae without septa, and C. implexum Nyl., which has bright
yellow thalli and a cottony texture.

Known from Neotropics and eastern Paleotropics (GBIF 2019, Rivas-Plata

& al, 2006, Uyenco 1963), Coenogonium botryosum was reported previously
for Veracruz, Mexico, growing on Ficus (Uyenco 1963); this is the first report
of the species on ferns.

Crocicreas quinqueseptatum S.E. Carp.,
Mem. New York Bot. Gard. 33: 160 (1981). Fics 11, 12
APOTHECIA cup-shaped, 0.5-0.8 mm diam. Disc white to yellowish
white (2A2, 2A1). MARGIN denticulate, inrolled over the hymenium when
dry, almost hyaline or translucent with long teeth; teeth fragile, formed by
groups of long, septate, hyaline hyphae, 100-115(-400 um) x 40-48 um,
lighter in colour than the rest of the apothecium, white to yellowish white
(2A1-2A2), 8-12 teeth per apothecium; breaking easily leaving the apothecia
non-denticulate. Stipe cylindrical, thin, concolorous with apothecia.
Ascli cylindrical, tapering toward the base, (60-—)80-105 x 6-8 um, thin-
walled, apical pore J+, biseriate, base without croziers. AscosporEs fusoid
to fusiform with subacute to acute ends, smooth, (20-)26-30 x 3-4 um
(Q = 5.5), 5-septate (rarely 6-7), hyaline. PARAPHYSES filiform, unbranched,

686 ... Medel-Ortiz, Baeza, Lorea-Hernandez

1-2 um diam., hyaline. EcraL ExcipuLum single layered, gelatinous,
composed of long cells in the inner part, extending outward from the disc to
form the teeth. MEDULLARY EXCIPULUM not differentiated.

Hasitat—On fallen stems and rachises of Alsophila firma and Cyathea
bicrenata.

SPECIMEN EXAMINED— MEXICO. VERACRUZ: San Andrés Tlalnelhuayocan County,

Los Encinales, 19°31.10’N 97°00.32’W, 1630 m, 10 August 2011, Medel & Lorea 2168.
Crocicreas quinqueseptatum was described by Carpenter (1981) as having
ascospores with a variable number (4-7) of septa and which are slightly
longer and narrower [(28—)30-33(-35) x (2-)2.5-3 um] than those found
in our specimen. Other features (outer apothecial surface, morphology,
and measurements) are close to C. helios var. parahelios S. E. Carp., which
differs by 0-3-septate ascospores, an absent or poorly developed medullary
excipulum, and broader apothecia (<1.5 mm; Carpenter 1981). Crocicreas
quinqueseptatum has been reported growing on angiosperm stems, especially
fallen herbaceous stems of Asteraceae in South America. This is the first
report of this species on ferns.

Cyathicula sp. Fics 8-10

APOTHECIA cup-shaped, sessile, disc flattened or concave, <1 mm diam.,
pastel yellow (3A4); margin dentate with 6-10 hyaline teeth per ascoma, teeth
composed of groups of long, hyaline, aseptate hyphae that extend beyond the
margin, each tooth separated by c. 420-480 um. Ascr cylindric-clavate, (96-)
110-140 x (7-9)10-11 um, 8-spored, biseriate, apical pore J-. ASCOSPORES
ellipsoidal-fusiform, 13-16 x 5-6 um (Q = 2.6), smooth, hyaline, 1-3-septate,
guttulate. PARAPHYSES filiform, apices simple or branched, 2-3 um diam. at
apex, septate; embedded with asci in a gelatinous matrix and separated only
with difficulty. ECrAL EXCIPULUM a textura prismatica to textura porrecta,
hyphae single-layered, long-celled, subparallel, close together, thick-walled,
hyaline, smooth, lacking crystals. MEDULLAR EXCIPULUM little differentiated,
hyphae 3-5 um diam., not easily observed due to gelatinous consistency.

Hasitat—On rachises of Cyathea bicrenata. Known only from our
collections.

SPECIMENS EXAMINED—MEXICO. VERACRUz: San Andrés Tlalnelhuayocan County;
E] Riachuelo, 19°30.99’N 97°00.39’W, 1626 m, 4 November 2011, Baeza 56; Los
Encinales, 19°31.10’N 97°00.32’W, 1630 m, 13 October 2011, Baeza 56a; 4 November
2011, Baeza 61, Castillo del Moral 64.

We place our material into Cyathicula based on its cupulate, sessile
apothecium with a toothed margin, whitish yellowish to pale brown color,

Pteridicolous ascomycetes (Mexico) ... 687

Lair , $10 pm [ 1mm

Figs 1-15. cf. Arachnopeziza. 1. Apothecia showing scarce subiculum and hairs (top right);
2. Ascospores in Congo red; 3. Excipulum showing hyphae from the subiculum. Bisporella
pteridicola. 4. Apothecia; 5. Asci with ascospores. Coenogonium botryosum. 6. Apothecia;
7. Ascospores. Cyathicula sp.; 8. Apothecia; 9. Cross section of excipulum, 10. One-septate
ascospores. Crocicreas quinqueseptatum. 11. Apothecia showing dentate margin; 12. Ascospores.
Dasyscyphella dryina. 13. Apothecium; 14. Ascospores. Durella macrospora. 15. Apothecia.

688 ... Medel-Ortiz, Baeza, Lorea-Hernandez

septate ascospores, J- asci, and the presence of a gelatinous matrix (Carpenter
& Dumont 1978b). Nonetheless, we must also note that the specimens fit
diagnostic characters of Crocicreas (Carpenter 1981). The controversy over
whether Crocicreas and Cyathicula are congeneric has lasted for decades
(Iturriaga & al. 1999, Zheng & Zhuang 2016). According to one classification
of Helotiales (Baral & al. 2015), Crocicreas is restricted only to the type species
C. gramineum (Fr.) Fr., and Cyathicula is unrelated. Although Wijayawardene
& al. (2018) support this distinction, we agree with the conclusion of Zheng
& Zhuang (2016) that existing data are insufficient for determining the
relationship between Crocicreas and Cyathicula. Molecular analysis is still
needed for us to assign our material definitively to a genus.

Dasyscyphella dryina (P. Karst.) Raitv.,
Akad. Nauk Estonskoi S.S.R., Inst. Zool. Bot., Tartu: 72 (1970). Fics 13, 14

APOTHECIA superficial, cup-shaped, stipitate, covered with white (1A1)
hairs. Disc 0.8-1 mm diam., yellowish white (2A2) to pale yellow (2A3),
smooth. STIPE cylindrical, covered with white (1A1) hairs with small hyaline
crystals at the tips. Harrs cylindrical, septate, slightly granular, hyaline,
easily visible, giving a distinctive villous appearance to the apothecium. AscI
cylindrical, 45-50 x 3-4 um, hyaline, 8-spored, irregularly biseriate, thin
walled, apical pore J+. Ascospores cylindric-ellipsoid to fusoid, (6-—)7-10
x 2-3 um, (Q = 4.2), smooth, hyaline, with rounded ends. PARAPHYSES
filiform, slightly lanceolate, 1 um diam., hyaline, projecting beyond the asci
by 10-20 um.

Hasitat—On rachises of Alsophila firma.

SPECIMENS EXAMINED—MEXICO. VeRAcRuz: San Andrés Tlalnelhuayocan

County, Rio Xocoyolapan, 19°30.89’N 97°00.39’W, 1590 m, 19 August 2011, Medel

2180, Medel 2181.
The studied specimen matches well with the concept of Lachnum
distinguendum sensu Dennis (1949, 1956, 1960), which is a synonym of
Dasyscyphella dryina. Dennis (1956) cited spores 2 um longer than those of
our collections, and filiform paraphyses which agreed with our specimen.
Rick (1906) described L. distinguendum Rick with broader apothecia
(<2 mm diam.), larger asci (50-69 x 7-10 um), larger ascospores (10-15 x
3-4 um), and lanceolate paraphyses (3-4 um diam.). Raitviir (1970) cited
slightly longer ascospores (9-12 x 2-3.2 um) and cylindrical paraphyses with
pointed tips. Nonetheless, our collection fits better Dennis's concept, because
our material has paraphyses that exceed the asci, while Raitviir (2002)
described paraphyses not exceeding the asci.

Pteridicolous ascomycetes (Mexico) ... 689

Dasyscyphella dryina is known from Europe and South America on Betula,
Fagus, and Populus (Dennis 1949, 1956. 1960, Raitviir 1970, Rick 1906).

Durella macrospora Fuckel,
Jahrb. Nassauischen Vereins Naturk. 23-24: 281 (1870). Fics 15-17

APOTHECIA superficial, cup-shaped, stipe short with a wide base. Disc
flat, 0.2-0.5 mm diam., wrinkled, brownish gray (10F2) with dark brown
(7F8) tones, hard consistency. Asci cylindrical (90-)100-130 x 11-12 um,
hyaline, 8-spored, irregularly biseriate, apical pore J-. Ascosporss ellipsoid-
fusiform, (18—)19-22(-23) x 4-5 um (Q = 4.2), smooth, hyaline, 3-septate,
constrained at the septum, ends rounded, with 4 small guttules. PARAPHYSES
cylindrical, slightly wider at the apex (c. 3 um) brownish to reddish brown in
KOH. EcTAL EXCIPULUM Of parallel brown hyphae.

Hasitat—On fronds of Sticherus palmatus.

SPECIMEN EXAMINED—MEXICO. VERACRUz: San Andrés Tlalnelhuayocan County,

El Riachuelo, 19°30.99’N 97°00.39’W, 1626 m, 8 December 2011, Medel 2210.
Spores of Durella macrospora have been cited as 15-21 x 3-5.5 um
(Breitenbach & Kranzlin 1984, Medardi 2004). Although the spores in
our collection are 2 um longer, other morphological characters (black
apothecia, hard consistency, ectal excipulum of parallel brown hyphae)
agree well with the published descriptions (Breitenbach & Kranzlin 1984,
Medardi 2004).

Durella macrospora has been previously recorded growing on wood of
deciduous trees such as Fagus, Malus domestica, and Quercus sp. (Breitenbach
& Kranzlin 1981, Medardi 2004), but not on ferns. Previously known only
from Europe (Breitenbach & Kranzlin 1984, Dennis 1956, Medardi 2004).

Hamatocanthoscypha helicotricha Huhtinen,
Karstenia 29(2): 182 (1990 [“1989”]). Fries 18-20

APOTHECIA cup-shaped, disc concave, pale yellow (3.A3) to pastel yellow
(3A4) shades, 0.5 mm diam., covered with fine, white (1A1) to yellowish
white (2A1-2A2) short hairs. Stipe cylindrical, short, slightly robust,
concolorous with apothecia. Hairs cylindrical, apically coiled to uncinate,
12-32 x 2-4 um, the apex tapering to <1 um diam., hyaline, septate
toward to the base, smooth. Ascr cylindric-clavate, apex conical, base
attenuate, (42—)45-72 x 6-7 um, hyaline, pore J-, 8-spored, biseriate, base
with croziers. Ascospores ellipsoid-fusoid, hyaline, smooth, 7-9(-10)
x 2-3 um (Q = 2.6), 1-septate, biguttulate. PARAPHysEs filiform, 2 um
diam., narrowing toward the base.

690 ... Medel-Ortiz, Baeza, Lorea-Hernandez

Hasitat—On rachises of Cyathea bicrenata and fronds of Sticherus
palmatus.

SPECIMENS EXAMINED—MEXICO. VeERAcRUZz: San Andrés Tlalnelhuayocan

County, El Riachuelo, 19°30.99’N 97°00.39’W, 1626 m, 8 December 2011, Medel &

Lorea 2213. Los Encinales, 19°31.10’N 97°00.32’W, 1630 m, 10 August 2011, Baeza

41,
Huhtinen (1990) and Raitviir (2004) report somewhat larger (9.8-12.8 x
2.8-4 um) spores than found in our specimens, but the presence of strongly
coiled hairs at the apex distinguishes this species. Hamatocanthoscypha
helicotricha has been recorded on fern rachises from Colombia and Cuba
(Huhtinen 1990, Raitviir 2004); this is the first report of the species for
Mexico.

Hyaloscypha fuckelii Nannf.,
Nova Acta Regiae Soc. Sci. Upsal., Ser. 4, 8(2): 273 (1932). Fics 21, 22

APOTHECIUM superficial, sessile, cup-shaped. Disc concave, 0.1-0.3 mm
diam., white (1A1), translucent when fresh or when rehydrated in KOH.
Receptacle with fine, whitish hairs. Harrs filiform, cylindrical, curved in
the apex, thin-walled, slightly granular. Ascr clavate, 50-66(-67) x 4-5
um, hyaline, thin-walled, 8-spored, irregularly biseriate, apical pore J+,
base with croziers. AscosporsEs ellipsoid, (5—)6-8(-10) x 2 um, (Q = 3.2),
0-1-septate, smooth, hyaline, with small oil drops. Parapuyses filiform,
protruding <9 um beyond asci, narrow, smooth, hyaline, apical cell with
small guttules.

Hasirat—On rachises of Alsophila firma.

SPECIMEN EXAMINED—MEXICO. VerRAcRUz: San Andrés Tlalnelhuayocan

County, El Riachuelo, 19°30.99’N 97°00.39’W, 1626 m,10 August 2011, Baeza 38.
Our collection agreed with Hyaloscypha fuckelii as described by Huhtinen
(1990). The similar H. albohyalina (P. Karst.) Boud. differs in its narrow
hairs and smaller spores.

Hyaloscypha fuckelii is widely distributed in Europe and North America
(Huhtinen 1990) and has been reported growing on monocotyledonous
genera such as Juncus, Typha, and Molinia. This is a new record for Mexico
and the first report on ferns.

Lachnum brevipilosum Baral,
Beih. Z. Mykol. 6: 74 (1985). Fics 23, 24

APOTHECIUM superficial, cup-shaped. Disc concave, white (1A1) to
pale (2A2), 0.8-1 mm diam. Receptacle covered with whitish hairs visible

Pteridicolous ascomycetes (Mexico) ... 691

to the naked eye. Stipe cylindrical, 0.6 mm long, covered by hairs generally
concolorous with disc (white, 1A1) except for some with a yellowish gray
(4B2) base. Hairs cylindrical, 130-150 x 4-5 um (the apex tapering to <2-3
um), 5-7-septate, hyaline, covered by granules. Asc clavate-cylindrical,
42-50 x 4-5 um, hyaline, pore J+, 8-spored, biseriate, croziers not observed.
Ascosporss ellipsoid, smooth, with rounded ends, 8-—10(-12) x 2 um (Q 4.2),
hyaline, 1-septate, biguttulate. PaRAPHysES filiform, septate, protruding by
5-10 um above the asci, hyaline.

Hasitat—On rachises of Alsophila firma and Cyathea bicrenata.
SPECIMENS EXAMINED—MEXICO. VERAcRuz: San Andrés Tlalnelhuayocan
County, Rio Xocoyolapan, 19°30.89’N 97°00.39’W, 1590 m, 4 November 2011,
Baeza 58. Paraje El Riachuelo, 19°30.99’N 97°00.39’W, 1626 m, 4 November 2011,
Cordova 86.

The Mexican specimens agree with the morphology of Lachnum
brevipilosum as provided by Dennis (1949, as Dasyscyphus brevipilus Le
Gal) and Ye & al. (2006), except for the length and width of the hair apices.

Lachnum brevipilosum has been reported growing on stems of Clematis,

Fagus, and Rubus from Europe and temperate China (Dennis 1949, Ye &
al. 2006) This is a new record for Mexico and represents the first report on
ferns.

Lachnum nudipes (Fuckel) Nannf.,
Svensk Bot. Tidskr. 22: 124 (1928). Fics 25, 26

APOTHECIUM superficial, cup shaped. Disc concave slightly flattened,
yellowish white (4A2) to pale yellow (4A3), 0.3-0.4 mm in diameter.
Receptacle densely covered with white (1A1) hairs. Stipe cylindrical, with
whitish hairs (1A1), more or less equal to the diameter of the disc. Harrs
cylindrical, ornamented with granules, widening at apex to 3-4 um, longer
than 80 um, hyaline in KOH. Asc clavate, (35-)38-47 x 4-5 um, 8-spored,
biseriate, pore J+, base without croziers. AscosporEs fusiform to elliptical
with rounded ends, (5—)6-8(-9) x 1-2(-3) um (Q = 3.1), smooth, hyaline.
PARAPHYSES lanceolate, septate towards the base, (50-)68-70 x 3-4 um,
clearly extending beyond the asci.

Hasitat—On fronds of Lophosoria quadripinnata.

SPECIMENS EXAMINED—MEXICO. VERACRUz: San Andrés Tlalnelhuayocan County,
Rio Xocoyolapan 19°30.89’N 97°00.39’W, 1500 m, 18 August 2011, Cérdova 72, 13
October 2011, Baeza 51; 4 November 2011, Cordova 65, 84, Castillo del Moral 65.
Our material agrees with Dennis's (1949) descriptions of Dasyscypha nudipes
(Fuckel) Sacc. var. nudipes and D. nudipes var. minor Dennis [both now treated

692 ... Medel-Ortiz, Baeza, Lorea-Hernandez

as synonyms of L. nudipes], but our ascomata and ascospore measurements
are closer to his D. nudipes var. minor.

Lachnum nudipes has been reported on Epilobium, Filipendula, and Spiraea
in Europe (Dennis 1949, as Dasyscypha). This is the first record Lachnum
nudipes for Mexico and the first report of its occurrence on ferns.

Lachnum pteridophyllum (Rodway) Spooner,
Biblioth. Mycol. 116: 470 (1987). Fics 27, 28

Macro and micromorphological characters agree with the description of
Medel & Lorea- Hernandez (2008).
Hasitat—On rachises of Cyathea divergens var. tuerckheimii, Cyathea
sp., Dryopteris sp., and Lophosoria quadripinnata.
SPECIMENS EXAMINED—MEXICO. VERACRUZ, San Andrés Tlalnelhuayocan
County, El Riachuelo, 13 October 2011, Baeza 29, 52, 53; 4 November 2011, Baeza
60, Medel 2196; 8 December 2011, Medel & Lorea 2214. Los Encinales, 19°31.10’N
97°00.32’W, 1630 m, 10 August 2011, Baeza 36. 19 August 2011, Medel 2179, 2182,
2185.
Lachnum pteridophyllum has been reported from Colombia, Dominican
Republic, Jamaica, Mexico, Panama, Peru, Puerto Rico, New Guinea, New
Zealand, Taiwan, Tasmania, and Venezuela (Haines 1980, Medel & Lorea-
Hernandez 2008). Haines (1980) cited this species on Cyathea sp. from
Oaxaca (Mexico), and Medel & Lorea-Hernandez (2008) from Veracruz on
decaying leaves of Dicksonia sellowiana, Cyathea divergens var. tuerckheimii,
and Cyathea spp. We add now two more hosts: Dryopteris sp. and Lophosoria
quadripinnata.

Lachnum singerianum (Dennis) W.Y. Zhuang & Zheng Wang,
Mycotaxon 67: 27 (1998). Fics 29, 30
Macro and micromorphological characters agree with the description of
Medel & Lorea- Hernandez (2008).
Hasitat—On rachises of Alsophila firma and Dicksonia sellowiana.

SPECIMEN EXAMINED—MEXICO. VERACRUz: San Andrés Tlalnelhuayocan County,

Rio Xocoyolapan, 19°30.89’N 97°00.39’W, 1590 m; 8 December 2011, Medel 2222.
Lachnum singerianum is known from the Andean region in South America
from Bolivia to Venezuela and from China, Jamaica, and Mexico (Haines
1980, Medel & Lorea-Hernandez 2008). Haines (1980) earlier reported
L. singerianum growing on rachises of ferns, while Medel & Lorea- Hernandez
(2008) reported the species on decaying leaves of Alsophila firma and
Dicksonia sellowiana in Veracruz.

Pteridicolous ascomycetes (Mexico) ... 693

|

20 um
-<¥ um

Fics 16-31. Durella macrospora. 16. Three-septate ascospores; 17. Asci with 1-septate ascospores.
Hamatocanthoscypha helicotricha. 18. Apothecia; 19. Asci and ascospores; 20. Uncinate or
coiled hairs. Hyaloscypha fuckelii. 21. Apothecia; 22. Ascospores. Lachnum brevipilosum.
23. Apothecium; 24. Septate hairs. Lachnum nudipes. 25. Apothecium (left) and ascospores (right);
26. Lanceolate paraphysis. Lachnum pteridophyllum. 27. Apothecium; 28. Ascospores. Lachnum
singerianum. 29. Apothecium; 30. Septate hair in 5% KOH. Lachnum varians. 31. Apothecia.

694 ... Medel-Ortiz, Baeza, Lorea-Hernandez

Lachnum varians (Rehm) M.P. Sharma,
Nova Hedwigia 43: 411 (1986). Fics 31, 32

Macro and micromorphological characters agree with the description of
Medel & Lorea- Hernandez (2008).

Hasitat—On rachises of Cyathea bicrenata and C. divergens var.
tuerckheimii and fronds of Adiantum andicola.

SPECIMENS EXAMINED—MEXICO. VeRAcRuz: San Andrés Tlalnelhuayocan

County, El Riachuelo, 19°30.99’N 97°00.39’W, 1626 m 18 August 2011, Baeza 47b,

Cordova 68, 69; 8 December 2011, Baeza 70.

ADDITIONAL SPECIMENS EXAMINED: MEXICO. VERACRUZ: Xalapa County,

Santuario del Bosque de Niebla, Parque Ecoldgico Francisco Javier Clavijero,

1 September 2013, Medel & Lorea 2259.
Lachnum varians is known from Australia, Hawaii, Mexico, New Guinea,
New Zealand, South America, and the Caribbean (Haines 1980, Medel
& Lorea-Hernandez 2008). The species was previously reported only on
decaying leaves of Alsophila firma (Medel & Lorea-Hernandez 2008);
the present study adds the new hosts Cyathea bicrenata, C. divergens var.
tuerckheimii, and Adiantum andicola.

Lachnum virgineum (Batsch) P. Karst.,
Bidrag Kannedom Finlands Natur Folk 19: 169 (1871). Fics 33, 34

APOTHECIA superficial, cupulate, 1-1.3 mm diam. Disc flat to concave,
white to yellowish white (1A1-1A2). Receptacle densely covered with
short, broad, white (1A1) hairs. Stipe, thin, covered with hairs concolorous
with the apothecia. Hairs cylindrical, 60 x 5-6(-8) um, granulose along
the entire length, 3-4-septate, the apex broadening <8 um diam. AscI
cylindric-clavate, 42-51x 4-5 um, 8-spored, biseriate, pore weakly blue
(J+). AScosPorgs fusiform, ends rounded, with one end sometimes wider
than the other, (6—)7-8 x 1-2 um (Q = 4.6). PaRAPHYSES lanceolate 4-5 um
diam., 2—3-septate, with small refractive guttules in the apex, extending
beyond the asci by 20 um.

Hasrirat—On fronds of Phlebodium aureum.

SPECIMENS EXAMINED—MEXICO. Veracruz: San Andrés Tlalnelhuayocan
County, El Riachuelo, 10 August 2011, Baeza 32; 8 December 2011, Medel 2208.
The Mexican specimens fit well the description of Lachnum virgineum
(Spooner 1987), especially the shape, color, and size of the apothecium
(densely covered by white hairs), spore size, and paraphyses exceeding the
asci by 15-20 um. Lachnum virgineum is similar to L. controversum (Cooke)
Rehm, which differs in obtuse or slightly swollen hair apexes and very slightly

Pteridicolous ascomycetes (Mexico) ... 695

narrower (3.5-4.5 um diam.) paraphyses. Although L. virgineum is generally
regarded as lignicolous, we found it growing on fronds of Phlebodium
aureum. Known from Australia and Europe (Breitenbach & Kranzlin 1984,
Spooner 1987).

Mollisia ventosa P. Karst.,
Bidrag Kannedom Finlands Natur Folk 19: 188 (1871). Fics 35-37
APOTHECIA superficial, sessile, discoid, flat, 0.5-1 mm diam., greenish-
yellow (1A7) with dark grey (1F1) to olive gray (1D2) margin. Receptacle
greenish gray (29F2), smooth. Ascr (72-)85-115 x 7-8(-10) um, apical
pore J+. Ascospores ellipsoid, (9-)12-15(-16) x 1.5-2 um (Q = 7.5),
1-3-septate, guttulate. PARAPHYSES filiform, slender, septate, branched or
not. ECTAL EXCIPULUM a textura angularis to textura globulosa, cells thick-
walled, brownish, 6-10(-12) x 6-10 um.
Hasrirat—On rachises of Cyathea bicrenata.
SPECIMENS EXAMINED—MEXICO. VeERaAcRuUz: San Andrés Tlalnelhuayocan
County, El Riachuelo, 19°30.99’N 97°00.39’W, 1626 m, 4 November 2011, Medel
2196; 8 December 2011, Baeza 71, Baeza 72.
Mollisia ventosa has been reported from Europe, and America (Breitenbach
& Kranzlin 1984, Dennis 1950, 1978, Medel & Chac6én 1997). Dennis (1950,
1978) described M. ventosa with spores larger (10-20 x 2-3.5 um) than
those in our Mexican collections, which instead match the measurements
cited by Breitenbach & Kranzlin (1984). They cited M. ventosa as growing on
Alnus and decorticated wood, but not on ferns. Mollisia undulatodepressula
(Feltgen) Le Gal & EF Mangenot, the only other Mollisia species known to
inhabit cloud forests in Mexico, grows on rotten wood (Medel & Chacén
1997).

Orbilia auricolor (A. Bloxam) Sacc.,
Syll. Fung. 8: 625 (1889). Figs 38, 39
APOTHECIA Superficial, scattered, sessile, cup-shaped. Discconcave,0.8-1mm
diam., yellow brownish to yellow gray (4B3-4B5) translucent when
rehydrated, margin crenulate. RECEPTACLE concolorous with disc, smooth.
Asct cylindric-clavate, 30-40(-42) x 2-4 um, hyaline, thin-walled, 8-spored,
biseriate, pore J—, base forked. Ascosporgs curved-filiform, (8—)9-10 x 1 um
(Q = 8.8), smooth, hyaline. PARAPHysEs cylindrical, apex capitate, with
incrustations, 3-4 um diam., septate, branched at base. ECTAL EXCIPULUM
textura globulosa with hyaline cells.

696 ... Medel-Ortiz, Baeza, Lorea-Hernandez

Hasitat—On rachises of Cyathea bicrenata and Marattia laxa.
SPECIMENS EXAMINED—MEXICO. VERACRUZ: San Andrés Tlalnelhuayocan
County, Rio Xocoyolapan 19°30.89’N 97°00.39’W, 1590 m, 8 December 2011, Baeza
67, Medel 2202.
The morphology of the apothecia, ascospores, asci, and paraphyses in our
Mexican specimens agree with Orbilia auricolor as described by Spooner
(1987) and Mo & al. (2005). The asexual stage of O. auricolor has been
recorded (as Arthrobotrys oligospora Fresen.) from Xochimilco, Mexico
(Chavarria & al. 2010).
Orbilia auricolor is known from Asia, Australia, Europe (Breitenbach
& Kranzlin 1984, Dennis 1981, GBIF 2019, Mo & al. 2005, Spooner 1987).
The species has previously been recorded growing on dead wood and
woody stems and other plant matter (Spooner 1987, Mo & al. 2005), but
not on ferns.

Protocreopsis pertusa (Pat.) Samuels & Rossman,
Stud. Mycol. 42: 66 (1999). FIGs 40, 41

PERITHECIA aggregated, globose, sessile, 0.8-1 mm diam., covered by
whitish (2A1) to yellowish white (3A2) hairs. Hairs cylindrical, curved
to wavy, smooth, thick-walled, hyaline, apices simple or forked, septate.
SuBICcULUM of thin whitish (2A1) hyphae, attached to the perithecia.
Asci clavate-cylindrical, 75-95(-100) x 10-12 um, thin-walled, hyaline,
ascospores irregularly biseriate, 8-spored, base with croziers. ASCOSPORES
ellipsoid, (13—)14-16 x 5 um (Q = 2.7), smooth, hyaline, 1-septate, with two
large guttules in the ends. PARAPHYSES filiform and hyaline.

Hasrrat—On rachises of Alsophila firma and Marattia laxa.

SPECIMENS EXAMINED—MEXICO. VeERAcRuz: San Andrés Tlalnelhuayocan

County, Rio Xocoyolapan, 19°30.89’N 97°00.39’W, 1590 m, 13 October 2011,

Cordova 77; 8 December 2011, Baeza 68, Medel & Lorea 2205.

ADDITIONAL SPECIMENS EXAMINED: MEXICO. VERAcRUz: San Andrés

Tlalnelhuayocan County, Rancho Agiiita Fria, 6 April 2007, Medel 1362.
Rossman & al. (1999) and Chaverri & al. (2010) described Protocreopsis
pertusa as having thin white mycelium beneath each perithecium,
thick-walled hairs densely united around the perithecial opening, and
1-3-septate spores measuring 13-17 x 4-5 um, all characters agreeing
with our Mexican specimens. This species has been reported from tropical
to subtropical regions on leaves of Musa, Heliconia, palms, bamboos, and
fern rachises. It is a new record for Mexico.

Pteridicolous ascomycetes (Mexico) ... 697

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Figs 32-48. Lachnum varians. 32. Asci with ascospores. Lachnum virgineum. 33. Apothecia;
34. Hair with enlarged apex. Mollisia ventosa. 35. Apothecia; 36. Excipulum at margin and lower
flank; 37. Asci with ascospores. Orbilia auricolor. 38. Apothecia 39. Paraphysis. Protocreopsis
pertusa. 40. Apothecia 41. Ascospores. Seimatosporium tostum. 42. Stromata 43. Asci with
ascospores, 44. Ascospores. Stictis carnea. 45. Apothecia. 46. Ascospores. Stictis radiata.
47. Apothecia; 48. Asci and ascospores.

698 ... Medel-Ortiz, Baeza, Lorea-Hernandez

Seimatosporium tostum (Berk. & Broome) Rossman & W.C. Allen, IMA Fungus
7(1): 5 (2016). Figs 42-44
StromartaA shield-like, growing within host tissue, black, grey (30F1) to
greenish grey (30F2) pigments are observed in KOH. Two perithecia per
stroma. Asctr cylindrical, 62-75 x 5-7(-8) um, thin-walled, apical pore
J+, spores 1-2-seriate, without croziers. Ascosporegs ellipsoidal-fusiform,
1-septate, constricted at the septum, smooth, poles subacute to acute,
(9-)10-11(-12) x 3-4 um (Q = 2.3). PARAPHYSES filiform, <1 um diam.,
extending beyond the ascus by 3-5 um, with tiny hyaline guttules at the
apex.
Hasitat—On rachises of Alsophila firma and Cyathea bicrenata.
SPECIMENS EXAMINED—MEXICO. VERACRUZ: San Andrés Tlalnelhuayocan County,
Rio Xocoyolapan, 19°30.89’N 97°00.39’W, 1590 m, 8 December 2011, Medel & Lorea
2108; 26 February 2001, Medel & Lorea 2201; Los Encinales, 19°31.10’N 97°00.32’W,
1630 m, 30 March 2014, Medel & Lorea 2379.
ADDITIONAL SPECIMENS EXAMINED: MEXICO. Veracruz: San Andrés
Tlalnelhuayocan, Rancho Agiiita Fria, 7 February 2007, Medel 1342.
Seimatosporium tostum is described by Paulus & al. (2006) as having larger
(>14 x 5 um) hyaline ascospores with only one septum, while Brockmann
(1976) described North American material (as Discostroma tostum) with
1-6-septate ascospores. The species has been reported growing on stems
of Epilobium. Known from North America (Brockmann 1976, Paulus & al.
2006), S. tostum establishes a new record for Mexico and the first report on
ferns.

Stictis carnea Seaver & Waterston,
Mycologia 33: 311 (1941). Fics 45, 46

APOTHECIA immersed in host tissue, hymenium greyish yellow to light
yellow (1A5-1B6), 05-0.7 mm diam., surrounded by an elliptical white
(1A1) ring. Ascr cylindric-clavate, 110-150 x 6-7 um, 8-spored, hyaline, J-.
Ascosporss long filiform, (80-)85-130(-150) x 3 um, tapering to one end,
multi-septate, twisted within the ascus. PARAPHYSES filiform, <3 um wide,
longer, hyaline, septate.

Hasitat—On rachises of Cyathea bicrenata.

SPECIMEN EXAMINED—MEXICO. VERACRUz: San Andrés Tlalnelhuayocan County;

Rio Xocoyolapan; 19°30.89’N 97°00.39’W, 1590 m, 4 November 2011, Medel 2112.
There are two species of Stictis growing on ferns in our study site, S. carnea
and S. radiata. ‘The differences in spore length and apothecial morphology
of the apothecia are sufficient to separate these two species (Sherwood 1977,

Pteridicolous ascomycetes (Mexico) ... 699

Johnston 1983). Stictis carnea has been reported from Asia, North America
(including Mexico), and New Zealand (Johnston 1983, Sherwood 1977),
growing on dead tissues of many herbaceous and woody plants and the
fern Cyathea medullaris (Johnston 1983). This species is a new report from
Mexico.

Stictis radiata (L.) Pers., Observ. Mycol. 2: 73 (1800 [“1799”]). Fics 47, 48

APOTHECIA deeply sunken in host tissue, hymenium vivid yellow to yellow
(3A7-3A8), <1 mm diam., surrounded by an irregular white (1A1) ring. Asc1
cylindric-clavate, 240-290 x (7-)10-12 um, 8-spored, hyaline, narrowed
toward the apex, pore J-. Ascospores long filiform, (219-)245-280(-283)
x 2-3 um, multiseptate, spirally arranged within the ascus. PARAPHYSES
filiform, 1 um diam., slightly widening (<2 1m) at the apex.

Hasitat—On _ rachises of Alsophila firma, Cyathea _ bicrenata,
Sphaeropteris horrida, and fronds of Polypodium sp.

SPECIMENS EXAMINED—MEXICO. VeERAcRuz: San Andrés Tlalnelhuayocan

County; Rio Xocoyolapan; 19°30.89’N 97°00.39’W, 1590 m, 4 November 2011,

Medel 2199; 8 December 2011, Baeza 69, Medel & Lorea 2200. Paraje El Riachuelo, 8

December 2011; Medel & Lorea 2207. Los Encinales, 19°31.10’N 97°00.32’W, 1630 m,

10 August 2011, Baeza 38b, Medel & Lorea 2169.
Diagnostic characters of Stictis radiata include ascomata with a deeply sunken
orange yellow hymenium and filiform multiseptate spores with interlaced
arrangement within the ascus and variably sized (180-325 x 1.9-2.8(-3) um;
Breitenbach & Kranzlin 1984, Johnston 1983, Sherwood 1977). Stictis radiata
resembles S. carnea but is distinguished by its shorter and wider ascospores.
Known from Mexico, New Zealand, and Australia (Johnston 1983, Sherwood
1977). In New Zealand, S. radiata has been reported on the ferns Cyathea
dealbata and Dicksonia squarrosa (Johnston 1983). We found S. radiata also
growing on Sphaeropteris horrida and Polypodium sp.

Discussion

Ferns, which comprise many genera and families, serve as habitat for a large
number of fungi. Our study indicates that most of the fungi on ferns belong
to Lachnaceae [previously included in Hyaloscyphaceae s.l.], represented by
eight species of Lachnum. Haines (1980), Samuels & Rogerson (1980), and
Medel & Lorea-Hernandez (2008) previously reported Hyaloscyphaceae s.l. on
ferns. Medel (2013), who conducted earlier research on Mexican cloud forest
ascomycetes, pointed out that Lachnaceae is “recurrent” on ferns in Cyatheaceae,
indicating that some of these fungi may live as endophytes in this fern family.

700 ... Medel-Ortiz, Baeza, Lorea-Hernandez

The dominant plant associates in this study belong to the “tree ferns” —
Alsophila firma, Cyathea bicrenata, C. divergens var. tuerckheimii, and
Sphaeropteris horrida. Herbaceous fern species with short stems at or
barely above ground level include Dryopteris sp., Lophosoria quadripinnata,
Marattia laxa, and ‘Thelypteris spp. Adiantum andicola and Sticherus
palmatus are classed as herbs with underground stems, while Grammitis
sp., Phlebodium aureum, Pecluma alfredii, and Polypodium sp., represent a
diverse epiphytic fern group. Two fern species hosted the highest number
of fungal species: Alsophila firma (11 species) and Cyathea bicrenata (12
species). The ascomycetes identified in this study grew more frequently on
petioles and rachises of tree ferns and some herbaceous ferns.

Our work stresses the importance of studying the highly diverse small
ascomycetes given that new records have been established by every study
(Braun 2004, Braun & al. 2013, Cannon 1997, Castaneda-Ruiz & Heredia
2000, Del Olmo & Arnolds 2014, Dingley 1972, Haines 1980, Kirschner
& Liu 2014, Medel & Lorea-Hernandez 2008, Samuels & Rogerson 1990,
Stevenson 1945); unfortunately, few have studied species from the Western
Hemisphere. This paper contributes new fern families and genera to the
list of ascomycetes hosts: Dicksoniaceae (genus Lophosoria), Marattiaceae
(Marattia), and Polypodiaceae (Grammitis and Pecluma). This is important
since there are few studies on the diversity of fungi on ferns, and most have
dealt only with genera of tree ferns in Cyatheaceae and Dicksoniaceae.

According to the literature surveyed, only seven ascomycete species
had previously been registered on ferns from Veracruz. This study adds 18
more records associated with ferns in Mexican cloud forests, bringing the

TABLE 1. Ascomycete fungi and fern hosts recorded from Mexico

SPECIES Host REFERENCE

HELOTIALES

Arachnopezizaceae

cf. Arachnopeziza Alsophila firma This paper
Dermataceae
Mollisia ventosa* Cyathea bicrenata This paper

Helotiaceae

Bisporella pteridicola* Alsophila firma, Cyathea bicrenata, This paper
Pecluma alfredii, Sphaeropteris horrida,
Thelypteris sp.

Crocicreas Cyathea bicrenata This paper

quinqueseptatum*

Crocicreas sessilis

Cyathicula sp.

Durella macrospora*
Hyaloscyphaceae

Dasyscyphella dryina*

Hamatocanthoscypha
helicotricha*

Hyaloscypha fuckelii*
Lachnaceae

Lachnum brevipilosum*

Lachnum fimbriiferum

Lachnum nudipes *

Lachnum oncospermatum

Lachnum pteridophyllum

Lachnum singerianum

Lachnum varians

Lachnum virgineum

Paradiopsidaceae
Dimeriella polypodii

HyPocrEALESs (Bionectriaceae)

Protocreopsis pertusa*
ORBILIALES (Orbiliaceae)

Orbilia auricolor*
OSTROPALES
Coenogoniaceae

Coenogonium botryosum
Stictidaceae

Stictis carnea*

Stictis radiata*

XYLARIALES (Sporocadaceae)

Seimatosporium tostum

Pteridicolous ascomycetes (Mexico) ... 701

Cyathea divergens var. tuerckheimii

Cyathea bicrenata

Sticherus palmatus

Alsophila firma

Cyathea bicrenata, Sticherus palmatus

Alsophila firma

Alsophila firma, Cyathea bicrenata

Alsophila firma, Cyathea bicrenata,
Cyatheaceae, Dicksonia sellowiana

Lophosoria quadripinnata

Dicksonia sellowiana

Cyathea divergens var. tuerckheimii,
Cyathea sp., Dicksonia sellowiana,
Dryopteris sp., Lophosoria
quadripinnata

Alsophila firma, Dicksonia sellowiana

Alsophila firma, Cyathea bicrenata,
C. divergens var. tuerckheimii,
Adiantum andicola

Phlebodium aureum

Polypodium montigenum, P. madrense

Alsophila firma, Marattia laxa

Cyathea bicrenata, Marattia laxa

Adiantum andicola, Grammitis sp.

Cyathea bicrenata

Alsophila firma, Cyathea bicrenata,

Sphaeropteris horrida, Polypodium sp.

Alsophila firma, Cyathea bicrenata.

Bold face indicates new hosts for the corresponding fungus

* indicates new fungal records for Mexico

Samuels & Rogerson
1990

This paper
This paper

This paper
This paper

This paper

This paper

Medel & Lorea-
Hernandez 2008

This paper
Medel & Lorea-
Hernandez 2008

Haines 1980; Medel &
Lorea-Hernandez 2008

Medel & Lorea-
Hernandez 2008

Haines 1980; Medel &
Lorea-Hernandez 2008

This paper

Samuels & Rogerson
1990

This paper

This paper

This paper

This paper
This paper

This paper

702 ... Medel-Ortiz, Baeza, Lorea-Hernandez

total to 25 ascomycete species distributed among Helotiales (20 species),
Hypocreales (1), Ostropales (3), and Xylariales (1). Taxonomic diversity of
Ascomycota reported in similar studies from other regions of the world show
a wide diversity of ascomycetes involved in biological interaction with ferns.
For example, Del Olmo & Arnolds (2014) reported the primary endophytes
to inhabit ferns in Costa Rica belonged to Dothideomycetes, Eurotiomycetes,
and Sordariomycetes, while mycosphaerellaceous fungi were more abundant
in Taiwan (Kirschner & Liu 2014) and other countries in Asia and Africa
(Braun & al. 2013). Only Haines (1980) cited Helotiales (Hyaloscyphaceae
s.l.) as the predominant fungi on tropical ferns, further supported by this
study where Lachnaceae was the most diverse family represented. To find
such diversity of ascomycetes associated with ferns, almost quadrupling the
number of species previously known for Mexico, suggests there are several
taxa yet to discover. We agree with Kirschner & Liu (2014) that research
on ascomycetes growing on ferns is still in its infancy. Additional work is
needed to establish the ecology of fern-associated fungi, particularly the
recurrence of some taxa on fern hosts.

Acknowledgments

The first author expresses her gratitude to Gast6n Guzman (+), who was a
constant guide during her studies. Thanks to DGI-Universidad Veracruzana for the
research assistant fellowship granted to Yajaira Baeza and to Juan Lara Carmona,
XAL mycological collection assistant, who processed loans during the our research.
Special thanks to Sharon Cantrell, Teresa Iturriaga, and Andrea I. Romero for
comments that greatly improved an early version of the manuscript. Luis Quijada
(Department of Organismic and Evolutionary Biology, Harvard Herbarium,
Cambridge MA, U.S.A.) and José Marmolejo (Facultad de Ciencias Forestales,
Universidad Autonoma de Nuevo Leon, México) made valuable suggestions during
their expert peer reviews. Special thanks to Shaun Pennycook for his exhaustive
revision.

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MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 707-717
https://doi.org/10.5248/134.707

First sexual morph record of Sarcopodium vanillae

NAPALAI CHAIWAN?”, SAJEEWA S.N. MAHARACHCHIKUMBURA},
DHANUSHKA N. WANASINGHE’, MINGKWAN DOILOM?’,
RUVISHIKA JAYAWARDENA’, KEVIN D. HyDE*””

"Center of Excellence in Fungal Research, Mae Fah Luang University,
Chiang Rai 57100, Thailand

? Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany,
Chinese Academy of Science, Kunming 650201, Yunnan, Peoples Republic of China

° School of Life Science and Technology, University of Electronic Science and Technology of China,
Chengdu 611731, Peoples Republic of China

* CORRESPONDENCE TO: kdhyde3@gmail.com

ABSTRACT—Sarcopodium vanillae was isolated from a dead leaf of Dracaena in Chiang
Rai Province, Thailand. Combined analyses of ACT, ITS, LSU, and TuB2 sequence data
obtained from the cultures derived from single spore isolates confirm that our collections
represent S. vanillae. This is the first record of the sexual morph, and the first record of S.
vanillae from Dracaena. A description and illustrations of both sexual and asexual stages of
S. vanillae are provided.

KEY worps—multigene, new host record, Nectriaceae, saprobe, taxonomy

Introduction

During our research on the diversity of microfungi in Thailand, we
recovered isolates from Dracaena in Chiang Rai Province that were typical
of Nectriaceae based on our preliminary morphological studies. Further
morphological and molecular characterization (multigene-based phylogeny
of nuclear ribosomal and protein-coding loci ACT, ITS, LSU and Tus2
sequence data) revealed the taxon as Sarcopodium vanillae. Nectriaceae
(Hypocreales; Rossman & al. 1999, Maharachchikumbura & al. 2015) is
typified by a characteristic perithecial wall structure and specific anamorphic

708 ... Chaiwan & al.

states. Its uniloculate perithecioid ascomata are yellow, orange, red, purple or
white, and the asexual morph is phialidic (Rogerson 1970, Rehner & Samuels
1995). The genus Sarcopodium Ehrenb. has had numerous nomenclatural
changes since it was first described in 1818 that have been synonymised
under Sarcopodium.

Cyphina Sacc., had already been synonymised under Sarcopodium by
Sutton (1977) and under “one fungus: one name” by Rossman & al. (2016)
supported the placement of the (asexual) Actinostilbe and the (sexual)
Lanatonectria under the much earlier (asexual) Sarcopodium. Sarcopodium
Ehrenb. is closely related to Pseudonectria Seaver. (Rossman & al. 1999).
Sutton (1981) synonymized Actinostilbe Petch. under Sarcopodium; and all
five species of Lanatonectria Samuels & Rossman (a genus described from a
sexual morph) have been transferred to Sarcopodium following phylogenetic
analyses (Lombard & al. 2015, Pennycook & Kirk 2019a). The new epithet for
the type species (“flocculentum”) has priority over the previous heterotypic
synonym, Sarcopodium macalpinei (Pennycook & Kirk 2019b).

Some Sarcopodium species have both sexual and asexual morphs, but S.
vanillae has been known only from its asexual morph (Sutton 1981; Rossman
1983, 1996, 2000; Rossman & al. 1993, 2013, 2016). This paper describes the
first record of a sexual morph for S. vanillae and reports Dracaena as a new
host record for this fungus.

Materials & methods

Sample collection, morphological study and isolation

Dead leaves of Dracaena were collected from Chiang Rai Province in Thailand
during November 2017. The samples were taken to the laboratory in Zip-lock bags.
Micro-morphological structures were observed and photographed with a Canon
EOS 600D digital camera fitted to a Nikon Eclipse Ni compound microscope. Single
spore isolates were obtained using the method described in Chomnunti & al. (2014).
Germinated spores were transferred to potato dextrose agar (PDA) plates and
incubated at 25-30 °C. Growth rates and culture characteristics were recorded after
three weeks. The morphological characters were measured using Tarosoft (R) Image
Frame Work v. 0.9.7 software. Figures were processed with an Adobe Photoshop CS6
Extended v. 10.0 software. The cultures are deposited in Mae Fah Luang University
Culture Collection, Chang Rai, Thailand (MFLUCC). Specimens are deposited in
the herbarium of Mae Fah Luang University, Chang Rai, Thailand (MFLU).

DNA extraction, PCR amplification and sequencing
Fungal isolates were grown on PDA for 3-4 weeks at 30 °C. The genomic DNA
was extracted from mycelium using the E.Z.N.A. Forensic DNA Kit. Amplification

Sexual morph of Sarcopodium vanillae (Thailand) ... 709

TABLE 1. Sequences from Pseudonectria and Sarcopodium species
(and Stachybotrys chartarum outgroup) used in the phylogenetic analysis

GENBANK ACCESSION NO.

TAXON CULTURE NO.
ACT ITS LSU TUB2

P. buxi CBS 324.53 KM231171 KM231778 KM231644 KM232037
P foliicola CBS 122566 KM231170 KM231777 KM231643 KM232036
S. circinatum CBS 587.92 KM231180 KM231787 KM231651 KM232046

CBS 100998 KM231179 KM231786 KM231650 KM232045
S. circinosetiferum CBS 100251 KM231175 KM231782 KM231646 KM232041
S. flavolanatum CBS 112283 KM231178 KM231785 KM231649 KM232044

CBS 128370 KM231177 KM231784 KM231648 KM232043
S. macalpinei CBS 115296 KM231176 KM231783 KM231647 KM232042
S. vanillae CBS 100582 KM231173 KM231780 HQ232174 KM232039

MEFLU 17-2595 MK692541 MK608516 MK691503 MK962543

MFLU 17-2597 MK692542 MK685870 MK691502 MK692544

Stachybotrys CBS 129.13 KM231268 KM231858 KM231738 KM232127
chartarum

New sequences are set in bold font.

primers used were: internal transcribed spacer (ITS)—ITS5 and ITS4 (White & al.
1990); 28S large subunit ribosomal RNA (LSU)—LROR and LRS5 (Rehner & Samuels
1994, Vilgalys & Hester 1990); partial actin gene (ACT)—ACT-512F and ACT-783R
(Carbone & Kohn 1999); partial beta-tubulin gene (ruB2)—Bt-2a and Bt-2b (Glass
& Donaldson 1995). The PCR mixer comprised 1 ul forward primer, 1 pul reverse
primer, 9.5 ul distilled deionized (DD) water, and 12.5 ul mixer. The PCR conditions
for ITS and LSU were 3 min at 94 °C; followed by 35 cycles of 30 s at 94 °C, 50 s at 55
°C, and 90 s at 72 °C; and a final elongation step at 72 °C for 10 min. Conditions for
ACT were an initial elongation step of 2 min at 95 °C; followed by 35 cycles of 45 s
at 95 °C, 45 s at 55 °C, and 1 min at 72 °C; and a final elongation step of 10 min at 72
°C. Conditions for TUB2 were an initial 8 min of 95 °C; followed by 35 cycles of 30 s
at 95 °C, 30 s at 55 °C, and 1 min at 72 °C; and a final elongation step at 72 °C for 5
min. The PCR products were purified and sequenced at Shanghai Sangon Biological
Engineering Technology and Service Co. Isolates including accession numbers of
gene sequences are listed in TABLE 1.

Phylogenetic analysis

Sequence data of Sarcopodium vanillae and related taxa (TABLE 1) were
downloaded from GenBank following Zeng & al. (2018) and Yang & al. (2018). The
multiple sequence alignments were produced with MAFFT v. 7 (http://maftft.cbre.jp/

710... Chaiwan & al.

alignment/server/index.html) and Biokdit v. 7.0.5.2 (Hall 1999). The phylogenetic
analyses were performed using maximum likelihood (ML) trees and generated using
the RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis & al. 2008, Stamatakis 2014) in
the CIPRES Science Gateway platform (Miller & al. 2010) using GTR+I+G model
of evolution. Parsimony analysis was carried out with the heuristic search option
in PAUP v. 4.0b10 (Swofford 2002) using the following parameters: characters
unordered with equal weight, random taxon addition, branch swapping with tree
bisection-reconnection (TBR) algorithm, branches collapsing if the maximum
branch length was zero. Alignment gaps were treated as missing characters in the
combined data set, where they occurred in relatively conserved regions. Trees were
inferred using the heuristic search option with 1000 random sequence additions,
with maxtrees set at 1000. Descriptive tree statistics for parsimony; tree length (TL),
consistency index (CI), retention index (RI), relative consistency index (RC) and
homoplasy index (HI) were calculated for trees generated. The Kishino-Hasegawa
tests (Kishino & Hasegawa 1989) were performed to determine whether trees
were significantly different. Bayesian analysis was conducted with MrBayes v. 3.1.2
(Huelsenbeck & Ronquist 2001) to evaluate posterior probability (PP) (Rannala &
Yang 1996) by Markov chain Monte Carlo sampling. GTR+I+G was used in the
command. Six simultaneous Markov chains were run for 2,000,000 generations with
trees sampled every 200th generation. The distribution of log-likelihood scores was
examined to determine stationary phase for each search and to decide if extra runs
were required to achieve convergence, using the program Tracer 1.4 (Rambaut &
Drummond 2007). First 10% of generated trees were discarded and remaining 90%
of trees were used to calculate posterior probabilities of the majority rule consensus
tree. The phylogenetic tree was figured in FigTree v. 1.4 (Rambaut 2014) and edited
using Microsoft Office Power Point 2007. Sequences derived in this study were
deposited in GenBank (TABLE 1).

Phylogenetic results

The combined sequence alignments comprised 12 isolates, with
Stachybotrys chartarum (CBS 129.13) as the outgroup taxon. The combined
dataset comprised 3065 characters including alignment gaps, of which 844
were derived from ITS, 624 from LSU, 812 from ACT, and 785 from TuB2.
The MP analysis for the combined dataset had 415 parsimony informative,
2400 constant, 250 parsimony uninformative characters and yielded a single
most parsimonious tree (TL = 1109, CI = 0.812, RI = 0.761, HI = 0.188,
RC=0.617; Fic 1). The RAxML analysis of the combined dataset yielded a
best scoring tree (Fic. 1) with a final ML optimization likelihood value of
-~9266.358949. The matrix had 656 distinct alignment patterns, with 17.28% of
undetermined characters or gaps. Estimated base frequencies were as follows:
A = 0.220970, C = 0.279441, G = 0.267083, T = 0.232506; substitution rates

Sexual morph of Sarcopodium vanillae (Thailand) ... 711

Pseudonectria follicola CBS122566
100/400/1

Pseudonectria buxi CBS324.53

Sarcopodium flavolanatum CBS 112283
100/101

Sarcopodium flavolanatum CBS128370

Sarcopodium circinatum CBS 100998
100/401

Sarcopodium circinatum CBS587.92

99/82/0.99

Sarcopodium circinosetiferum CBS100251
100/100/1

Sarcopodium macalpinel CBS115296

Sarcopodium vanilla CBS100582
100/101

64/100/0.95

Fic. 1. Phylogram generated from RAxML based on combined ITS, LSU, ACT, and Tus2 sequence
data. Bootstrap support values for maximum likelihood (ML) 260%, maximum parsimony (MP)
260%, and Bayesian posterior probabilities (PP) 20.95 are indicated as ML/MP/PP at the nodes.
Ex-type strains are in bold and the strains from this study are indicated in green.

712 ... Chaiwan & al.

AC = 0.730175, AG = 1.879094, AT = 1.313827, CG = 0.798328, CT = 4.086170,
GT = 1.000000; gamma distribution shape parameter a = 0.147281.

Bayesian posterior probabilities from Bayesian inference analysis were
assessed with a final average standard deviation of split frequencies = 0.0056.
The phylogenetic tree in this study showed that our strains (Sarcopodium
vanillae MFLUCC 17-2595 and MFLUCC 17-2597) grouped in the
Sarcopodium clade, and formed a well-supported cluster with S. vanillae
(CBS 100582) with 64% ML, 100% MP, and 0.95 PP.

Taxonomy

Sarcopodium vanillae (Petch) B. Sutton,
Trans. Brit. Mycol. Soc. 76: 99 (1981) PLATES 1, 2
= Actinostilbe vanillae Petch, Ann. Roy. Bot. Gard. (Peradeniya) 9(3):327 (1925)

SAPROBIC on dead leaves of Dracaena (Asparagaceae).

SEXUAL MORPH: ASCOMATA 150-200 um high, 160-240 um wide (x = 173
x 189 um, n = 5), perithecial, subglobose, solitary or in groups, soft-textured,
pale yellow or rarely orange, superficial on a leaf or erumpent, with a papillate
ostiole. PERIDIUM 15-25 um wide (x = 47 um, n = 5), composed of several
layers of white to light orange cells of textura angularis. Asci 36-52 x 3-5 um
(x = 44 x 4.5 um, n = 20), 4-spored, unitunicate, cylindrical, rounded at apex,
clavate to fusiform, short pedicellate. Ascosporss 8-12 x 3-4.5 um (x = 11
x 3.9 um, n = 20), fasciculate, broadly elongate, 1-septate.

ASEXUAL MORPH: Myce.Lium, branched, septate, hyaline, smooth.
CONIDIOMATA 200-210 x 220-240 um (x= 205 x 230 um, n= 5), sporodochial,
solitary or gregarious, setiferous, yellow to bright yellow or rarely orangish
brown, soft-textured, superficial, separate, gregarious or confluent, sessile,
attached to the substratum by a small stroma concentrated in the epidermis
or outer layers of peridermal tissue, pulvinate, setose. SETAE 100-110 x 5-10
um (x = 105 x 7.5 um, n = 5), septate, unbranched, cylindrical, incurved,
erect, very thick-walled, medium to pale golden brown, more or less straight
(occasionally slightly bent either at the base or nearer the apex), erect,
hyaline, pointed or rounded at the apex. CONIDIOPHORES mononematous,
verticillately or penicillately branched, straight or flexuous, smooth, hyaline,
short, septate, with 1-4 monochasial branching, compactly arranged,
cylindrical, intermixed with long setae. CONIDIOGENOUS cells enteroblastic,
monophialidic, integrated, cylindrical or more frequently tapered towards
the apices, subulate, widest from middle to base, 10-20 x 1.2-2.7 um
(x = 15 x 1.95 um, n = 5), with inconspicuous collarette, hyaline, smooth,

Sexual morph of Sarcopodium vanillae (Thailand) ... 713

PLATE 1. Sarcopodium vanillae (MFLU 19-0567, herbarium specimen): A, B. Perithecia on
host surface; C-E. Perithecial cross sections; F Crush mount of perithecium; G. Conidioma on
perithecium; H, I. Asci, J. Ascospores; K, L. PDA cultures. Scale bars: A, B = 200 um; C-G = 50 um;
H, I= 20 um; J = 10 um

formed as the ultimate branches of conidiophores and completely covering the
external face of the conidiomata. Conrip1a 5-9 x 2.1-2.6 um (x = 6 x 2.5 um,
n = 20), cylindrical, 0-1-septate, smooth, hyaline, rounded at both ends, held
together in a slimy mass, ellipsoid to oval, straight.

CULTURE CHARACTERISTICS: Colony on PDA reaching 30-40 mm diam.
after 3 weeks at 25-30 °C; from above, white to yellow at margin, white to

714 ... Chaiwan & al.

PLATE 2. Sarcopodium vanillae (MFLU 19-0566, herbarium specimen): A-C. Conidiomata on host
surface; D. Conidioma; E-I. Setae; J, K. Setal bases; L. Setal apex; M. Conidia; N, O. PDA cultures.
Scale bars: A-C = 200 um; D, J, K = 100 um; E-I, L, M = 50 um.

orange in the middle, white at centre; from below, yellow, medium dense,
irregular, slightly raised to umbonate, surface slightly rough, dull with
umbonate edge, concave at centre, fluffy to floccose, with white tufts at centre.

SPECIMENS EXAMINED: THAILAND, CHIANG RAI PROVINCE, Mae Lao District, on
dead leaf of Dracaena, 17 November 2017, Napalai Chaiwan NCCR003 (MFLU 19-
0566 [asexual morph]; living culture MFLUCC 17-2595); Napalai Chaiwan NCCR004
(MFLU 19-0567 [sexual + asexual morph]; living culture MFLUCC 17-2597).

Discussion

Our strains share similar morphological characters with S. vanillae strain
CBS 100852, which was isolated from Anthurium sp. in Ecuador (Lombard &
al. 2015). The phylogenetic analysis also supports the close relationship. The
genus Sarcopodium has both sexual and asexual morphs (Wijayawardene
& al. 2017a,b, 2018). The conidial morphology of our strain is similar to
S. circinatum (the type species of the genus); however our conidiomata
and conidiophores more closely resemble Volutella ciliata (CBS 483.61)

Sexual morph of Sarcopodium vanillae (Thailand) ...715

(Lombard & al. 2015). Previously, only the asexual morph has been observed
for S. vanillae (Sutton 1981), and this study is the first report of the sexual
morph. Sarcopodium vanillae has been reported from Abelmoschus manihot
in Papua New Guinea, Citrus nobilis in Brunei, Vanilla planifolia in Sri Lanka,
and V. tahitensis in Papua New Guinea (Farr & Rossman 2019). This study
provides the first report of S. vanillae from Dracaena, and its first report from
Thailand.

Acknowledgments

N. Chaiwan thanks the Thailand Research Fund (PHD60K0147) and Kunming
Institute of Botany for financial support and the molecular laboratory for support.
K.D. Hyde thanks the grants entitled: 1 the future of specialist fungi in a changing
climate: baseline data for generalist and specialist fungi associated with ants,
Rhododendron species and Dracaena species (Grant number: DBG6080013) and
2. The climate changes grant: Impact of climate change on fungal diversity and
biogeography in the Greater Mekong Subregion (Grant number: RDG613001).
M. Doilom thanks the 5th batch of Postdoctoral Orientation Training Personnel in
Yunnan Province and the 64th batch of China Postdoctoral Science Foundation.
D.N. Wanasinghe thanks the CAS President’s International Fellowship Initiative
(PIFI) for funding his postdoctoral research (number 2019PC0008), the National
Science Foundation of China and the Chinese Academy of Sciences for financial
support under the following grants:41761144055, 41771063 and Y4ZK111B01.
S.C. Karunarathna thanks the CAS President's International Fellowship Initiative
(PIFI) for funding his postdoctoral research (number 2018PC0006). The National
Science Foundation of China (NSFC) for funding this work under the project code
31750110478. Jianchu Xu thanks the Key Research Program of Frontier Sciences
“Response of Asian mountain ecosystems to global change’, CAS, Grant No.
QYZDY-SSW-SMC014. All authors are grateful to peer experts D. Jayarama Bhat
(Emeritus Professor of Botany. Goa University, India) and Eric H.C. McKenzie
(Manaaki Whenua-Landcare Research, Auckland, New Zealand) for their assistance
and presubmission review.

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MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 719-730
https://doi.org/10.5248/134.719

Notes on rust fungi in China 7. Aecidium caulophylli
life cycle inferred from phylogenetic evidence and
renamed as Puccinia caulophylli comb. nov.

JING-XIN Jr'4, ZHUANG LY, Yu L1’, MAKOTO KAKISHIMA”?4®

' Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi,
Jilin Agricultural University, Changchun, Jilin 130118, China

College of Plant Pathology, Shandong Agricultural University, Taian 271000, China

> College of Forestry, Beijing Forestry University, Beijing 100083, China

‘University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

* CORRESPONDENCE TO: *1096314395@qq.com * kakishima.makoto.ga@u.tsukuba.ac.jp

ABSTRACT—Spermogonial and aecial stages of Aecidium caulophylli on Caulophyllum
robustum (Berberidaceae) were shown by phylogenetic analyses of ITS and 28S sequence data
to be identical to a uredinial and telial rust on Milium effusum (Poaceae). A new combination,
Puccinia caulophylli is proposed for this species, and an epitype is designated.

Key worps—Pucciniaceae, Pucciniales, taxonomy

Introduction

Aecidium caulophylli was described in 1898 based on spermogonial and

aecial stages of a specimen on Caulophyllum robustum (Berberidaceae)

collected by V. Komarov in Amur, Siberia, Russian Far East, during June
1895 (Saccardo & Sydow 1902). This rust has been reported from China,
Russian Far East, and Japan (Miura 1928, Ito 1950, Tai 1979, Harada 1984,
Hiratsuka & al. 1992, Azbukina 2005). However, its uredinial and telial stages
have not been clearly demonstrated, although Azbukina (1984, 2005) listed
this species as spermogonial and aecial stages of Puccinia brachypodii var.
poae-nemoralis (G.H. Otth) Cummins & H.C. Greene [= P. poae-nemoralis

G.H. Otth], without any evidence.

ji & al.

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Puccinia caulophylli comb. nov. (China) ... 721

During an investigation of rust fungi in Jilin Province, China, during
2015-2018, A. caulophylli was observed on C. robustum. Near the infected
C. robustum, uredinial and telial stages of Puccinia species were also
observed on plants of Poaceae and Cyperaceae. We suspected that this rust
may have an heteromacrocyclic life cycle, producing uredinia and telia
on one of these plants. Inoculation of plants with spores is an appropriate
method to clarify rust fungus life cycles (Ji & al. 2017a,b), but often plant
collection may be restricted in conservation areas. As it may be difficult
to supply appropriate growth conditions after transplantation, molecular
analyses have been applied to clarify rust life cycles (Liu & Hambleton
2013, Ji & al. 2016, Padamsee & McKenzie 2017, Scholler & al. 2019). We
report here the results of phylogenetic and morphological analyses using
spermogonial/aecial specimens on A. caulophylli and potentially related
uredinial/telial specimens on Poaceae and Cyperaceae.

Materials & methods

Molecular analyses

Spermogonial (0) and aecial (I) stages of A. caulophylli on C. robustum were
collected in Jiaohe, Jilin Province, China, and used for molecular analyses. Rust
specimens on Milium effusum (Poaceae) were collected in several areas in Jilin and
Heilongjiang Provinces, China, as preliminary phylogenetic analyses suggested they
represented the uredinial (II) and telial (III) stages of A. caulophylli. The specimens
were collected during surveys of rust fungi in Jilin Province from 2015 to 2018. For
comparative analyses, sequences were included from two life cycles that previously
clarified by inoculations (Ji & al. 2017a,b): (1) Puccinia klugkistiana (Dietel) Jing
X. Ji & Kakish. on Ligustrum obtusifolium Siebold & Zucc. (0, I) and Cleistogenes
hackelii (Honda) Honda (II, II), collected in Changchun, Jilin Province; and
(2) PB adenocauli (Syd. & P. Syd.) Jing X. Ji & Kakish. on Adenocaulon himalaicum
Edgew. (0, I) and Carex onoei Franch. & Sav. (II, IID).

Total genomic DNA was directly extracted from about 200 spores obtained from
single sori on the leaves of each specimen, using similar methods reported by Ji & al.
(2016, 2019). Specimens used in the experiments were deposited in the Herbarium
of Mycology, Engineering Research Center of Chinese Ministry of Education for
Edible and Medicinal Fungi, Jilin Agricultural University, China (HMJAU) and all
data sequenced in this experiment were deposited at GenBank (TABLE 1).

Sequences from the specimens were aligned following Ji & al. (2019). ITS and
28S sequence data retrieved from GenBank were added to phylogenetic analyses.
Accession numbers for these data are shown in the phylogenetic trees (Fics 1,
2). Phylogenetic trees were constructed with the sequences of Gymnosporangium
yamadae Miyabe ex G. Yamada and G. asiaticum Miyabe ex G. Yamada as outgroup,

722... Ji &al.

Puccinia caulophylli HMJAU8620 [Caulophyllum robustum] (SA)
Puccinia caulophylli HMJAU8619 [Milium effusum)] (UT)
Puccinia caulophylli HMJAU8623 [Milium effusum] (UT)
Puccinia caulophylli HMJAU8618 [Milium effusum] (UT)
Puccinia caulophylli HMJAU8625 [Milium effusum] (UT)
Puccinia caulophylli HMJAU8626 [Milium effusum] (UT)
059/50" Puccinia caulophylli HMJAU8621 [Milium effusum)] (UT)
Puccinia caulophylli HMJAU8627 [Milium effusum] (01)
Puccinia caulophylli HMJAU8534 [Caulophyllum robustum] (SA)
Puccinia caulophylli HMJAU8622 [Milium effusum] (U1)
Puccinia caulophylli HMJAU8624 [Caulophyllum robustum] (SA)
Puccinia caulophylli HMJAU8531 [Caulophyllum robustum] (SA)
osoisyg) -Puecinia caulophylli HMJAU8532 [Caulophyllum robustum) (SA)
Puccinia sessilis (AY 217134)
Puccinia sessilis (AY 217135)
Puccinia hordei (KY764128) [Ornithogahim arabicum]
Puccinia recondita (KY798399) [Elymus spicatus]
wioonooPuecinia magnusiana (GU058000) [Phragmites sp.]
Puccinia magnusiana (KY 764139) [Phragmites sp.]
Puccinia triticina (DQ664194) [Triticum aestivum]
Puccinia triticina (KY764169) [Triticum aestivum]
Puccinia recondita (DQ417424) [Aegilops ovata]
‘Puccinia recondita (AF511082)
Puccinia recondita f. sp. secalis (DQ417423) [Secale cereale]
Puccinia clavata (KX985761) [Clematis foetida]
Puecinia hordei (KX985762) [Holcus lanatius]
osgousgPuccinia sorghi (KY764162) [Zea mays]
Puccinia sorghi (GU057994) [Zea sp.]
Puccina polysora (GU058024) [Zea mays]
'Puccina polysora (MF033480) [Zea sp.]
‘Puccinia brachypodii (KX999868) [Poa annua]
‘Puccinia poae-nemoralis (KY 798384) [Calamagrostis sp.]
oossiggjPuccinia coronata (AF426207) [Rhamnus cathartica]
'Puccinia coronata (EU851141) [Holcus lanatus]

4 Puccinia coronata (DQ355448) [Bromus sp.]
Puccinia coronata (AB693935)
0.95/°/73 Puccinia graminis (KM249852) [Glyceria maxima]

oseomeyPuccinia graminis (KY798389) [Poa annua]
'Puccinia graminis (AF522177)
Puccinia adenocauli HMJAU 8628 [Adenocaulon himalaicum] (SA)
nooo Puccinia adenocauli HMJAU 8630 [Carex onoei] (UT)
Puccinia adenocauli HMJAU 8274 [Carex onoei] (UT)
Puccinia adenocauli HMJAU 8629 [Adenocaulon himalaicum] (SA)
Puccinia klugkistiana HMJAU 8280 [Cleistogenes hackelii] (U1)
Puccinia klugkistiana HMJAU 8633 [Ligustrum obtusifolium] (SA)
soon oo| Puccinia klugkistiana HMJAU 8632 [Cleistogenes hackelii] (UT)
Puccinia klugkistiana HMJAU 8198 [Ligustrum obtusifolium] (SA)
Puccinia klugkistiana HMJAU 8282 [Cleistogenes hackelii] (UT)
Puccinia klugkistiana HMJAU 8631 [Ligustrum obtusifolium] (SA)
/100/100) Gymnosporangium asiaticum HMJAU 8324 [Pyrus sp.]
Gymnosporangium yamadae HMJAU 8096 [Malus baccata]

1/100/100)

Fic. 1. Phylogenetic tree constructed by MP method based on sequences of 28S regions of rDNA.
Bootstrap values of MP and ML are followed by the Bayesian posterior probabilities (BPP) on
the nodes in the topology. Asterisk (*) represents bootstrap values <50% or BPP <0.5 in the
topology. Sample data are shown with species name, voucher specimen number or GenBank
accession number (in parentheses), and host plant. Sequence data determined in this study are
shown in bold face. SA: Spermogonial and aecial stages, UT: Uredinial and telial stages.

according to Ji & al. (2019). The alignment and trees were deposited in TreeBase
under http://purl.org/phylo/treebase/phylows/study/TB2:S24307 (Fic. 1) and
TB2:824308 (Fic. 2).

Morphological observations

Light (LM) and scanning electron (SEM) microscopy were used to examine
morphological characters of rust specimens including the size and shape of sori and
spores following Ji & al. (2019).

Puccinia caulophylli comb. nov. (China) ... 723

Puccinia caulophylli HMJAU8620 [Caulophyllum robustum] (SA)
Puccinia caulophylli HMJAU8619 [Milium effusum] (U1)
Puccinia caulophylli HMJAU8618 [Milium effusum] (UT)
Puccinia caulophylli HMJAU8532 [Caulophyllum robustum] (SA)
Puccinia caulophylli HMJAU8534 [Caulophyllum robustum] (SA)
Puccinia caulophylli HMJAU8625 [Milium effusum] (U1)
Puccinia caulophylli HMJAU8627 [Milium effusum] (UT)
Puccinia caulophylli HMJAU8623 [Milium effusum] (UT)
Puccinia caulophylli HMJAU8626 [Milium effusum) (U1)
vosioo| Puccinia caulophylli HMJAU8531 [Caulophyllum robustum] (SA)
Puccinia caulophylli HMJAU8621 [Milium effusum] (UT)
99/55/62] Puccinia caulophylli HMJAU8624 [Caulophyllum robustum] (SA)
Puccinia caulophylli HMJAU8622 [Milium effusum] (UT)
Puccinia sessilis (AY 217134)
1100/1001 Pyecinia sessilis (AY 217135)
joanoor Puccinia triticina (DQ417417) [Triticum turgidum L. var. durum |
o.p/ST/EM Puccinia triticina (DQ417417) [Triticum sp.]
Puccinia triticina (KT982695) [Hordeum vulgare]
1007108 Puccinia triticina (DQ460717) [Hordeum vulgare]
Puccinia recondita f. sp. secalis (DQ417426) [Secale cereale]
M0000 Puccinia recondita (AY956562) [Cerinthe minor]
0.87/#/4 Puccinia coronata (DQ355444) [Holcus lanatus|
0.99/88 Puccinia coronata f. sp. graminicola (HM131240) [Arrhenatherum elatius]
Puccinia coronata var. coronata (HM057141) [Calamagrostis epigejos]
Puccinia coronata f. sp. avenae (EU014044) [Lolium perenne]
Puccinia coronati-hordei (HM131229) [Elymus repens]
Puccinia coronati-japonica (HM131317) [Calamagrostis arundinacea]
Puccinia coronati-agrostidis (HM131319) [Agrostis stolonifera]
Puccinia coronati-calamagrostidis (HM131350) [Elymus sp.]
venoqPuccinia sorghi (AY 114291)
Puccinia sorghi (HQ154038) [Zea mays]
vioggg Puccinia brachypodii (KM 391664)
i600} | Puccinia brachypodii (KM 391669)
Puccinia brachypodii (GQ457303)
Puccinia graminis (AY874140)
oom Puccinia graminis f. sp. tritici (DQ 417379)
Puccinia graminis (HM131357) [Elymus repens]
Puccinia klugkistiana HMJAU 8633 [Ligustrum obtusifolium] (SA)
Puccinia klugkistiana HMJAU 8282 [Cleistogenes hackelii] (UT)
Puccinia klugkistiana HMJAU 8198 [Ligustrum obtusifolium] (SA)
Puccinia klugkistiana HMJAU 8631 [Ligustrum obtusifolium] (SA)
sno Puccinia klugkistiana HMJAU 8632 [Cleistogenes hackelii| (UT)
0.98/52/8 Puccinia klugkistiana HMJAU 8280 [Cleistogenes hackelii| (UT)
oonoor Puccinia polysora (HQ189433) [Zea mays]
Puccinia polysora (HM467909)
Puccinia adenocauli HMJAU 8274 [Carex onoei] (UT)
Puccinia adenocauli HMJAU 8628 [Adenocaulon himalaicum] (SA)
1100/1001 '— Puccinia adenocauli HMJAU 8629 [Adenocaulon himalaicum] (SA)
Puccinia adenocauli HMJAU 8630 [Carex onoei] (UT)
1/100/100 Gymnosporangium asiaticum HMJAU 8324 [Pyrus sp.]
Gymnosporangium yamadae HMJAU 8096 [Malus baccata]

/54/84)

99}

0.54/52/56

0,98/*/67
/97/100

0.97738763)

0.98/*/86)

+/#/8;

/1.00/100

Fic. 2. Phylogenetic tree constructed by MP method based on ITS regions of rDNA. Bootstrap
values of MP and ML are followed by the Bayesian posterior probabilities (BPP) on the nodes in
the topology. Asterisk (*) represents bootstrap values <50% or BPP <0.5 in the topology. Sample
data are shown with species name, voucher specimen number or GenBank accession number
(in parentheses), and host plant. Sequence data determined in this study are shown in bold face.
SA: Spermogonial and aecial stages, UT: Uredinial and telial stages

Results & discussion

Phylogeny and life cycle

The 28S dataset comprised 51 sequences of 50 taxa with 525 total
characters, including 430 constant characters, 25 parsimony-uninformative
variable characters, and 70 parsimony-informative characters. Parsimony
analysis yielded one parsimonious tree with TL = 159, CI = 0.679, RI = 0.864

724 ... Ji & al.

and RC = 0.587. Bayesian analysis resulted in average standard deviation of
split frequencies of 0.005869. The final ITS dataset comprised 51 sequences
of 50 taxa with 820 total characters, of which 336 were parsimony-
informative. Parsimony analysis yielded one parsimonious tree with
TL=995, CI = 0.623, RI = 0.840 and RC = 0.523. Bayesian analysis resulted in
average standard deviation of split frequencies of 0.004251. Tree topologies
formed by MP, ML, and MCMC methods were identical among trees.
The phylogenetic trees generated through Bayesian analysis are shown in
Fiac..1 (28S) and! Fie. 2(ITS):

Both 28S and ITS phylogenetic trees placed spermogonial and aecial
stages on C. robustum (HMJAU 8531, 8532, 8534, 8620, 8624) and uredinial
and telial stages on M. effusum (HMJAU 8618, 8619, 8621, 8622, 8623,
8625, 8626, 8627) within a monophyletic clade (Fries 1, 2). All stages of
P. klugkistiana and P adenocauli were placed in separate monophyletic
clades, confirming that heteroecious life cycles of rust fungi can be revealed
by phylogenetic analyses as demonstrated by Liu & Hambleton (2013),
Padamsee & McKenzie (2017), and Scholler & al. (2019). The phylogenetic
analyses also supported the life cycle connection between the rust on
C. robustum and the rust on M. effusum.

Although the Caulophyllum/Milium rust was shown to be phylogenetically
close to P. sessilis W.G. Schneid. ex J. Schrot., P. triticina Erickss., and
P. recondita Roberge ex Desm., these three rusts inhabit different host
plants. This rust is also genetically distant from P brachypodii G.H. Otth and
P. poae-nemoralis, two other telial rusts reported on Milium spp. (Cummins
1971, Zhuang & al. 1998, Azbukina 2005). Therefore, we conclude that this
rust is distinct from other species.

Morphology & taxonomy

From the phylogenetic analyses, the spermogonia and aecia on
A. caulophylli and the uredinia and telia on M. effusum are produced
by one and the same heteromacrocyclic rust species. LM and SEM
observations showed that overall morphology of the rust on C. robustum
is identical with that of A. caulophylli described by Saccardo & Sydow
(1902), Ito (1950), Harada (1984), and Hiratsuka & al. (1992) (Fics 3,
54,B). The morphologies of uredinial and telial specimens on M. effusum
were similar to each other (Fics 4, 5c-£), and the two-celled teliospores
refer the rust to the genus Puccinia (Cummins & Hiratsuka 2003).

Specimens on Milium effusum are phylogenetically close to P. sessilis,
but urediniospores on M. effusum are bigger than those of P. sessilis (20-32

Puccinia caulophylli comb. nov. (China) ... 725

ae

>

AS

ye ey
S ay, 4 io

FAM
ae

Fic. 3. Puccinia caulophylli on Caulophyllum robustum: spermogonial and aecial stages.
A. Yellow lesions on the leaves producing spermogonia and aecia; B. Aecia produced
around spermogonia on lower surface of the plant; C. Catenulate aeciospores surrounded
by peridium in a vertical section of an aecium; D. Vertical section of a spermogonium;
E. Aeciospores. Scale bars: C, D = 30 um; E = 15 um.

x 19-25 um), and teliospores are smaller than those of P. sessilis (32-58 x
13-20 um). Additionally, the rust on M. effusum has uredinial paraphyses
that have not been reported in P. sessilis (Hiratsuka & al. 1992, Zhuang & al.
1998). PB. brachypodii var. poae-nemoralis occurs on M. effusum (Cummins
1971, Zhuang & al. 1998) and its telial structures and teliospores are

726 ... Ji & al.

morphologically similar to the present rust fungus. However, urediniospores
of the current rust are bigger than those of P. brachypodii var. poae-nemoralis
(18-25 x 15-23 um). The aecial stage of P brachypodii var. poae-nemoralis
occurs on Berberis spp. (Cummins 1971).

Aecidium caulophylli is a legitimate name under the INTERNATIONAL CODE
OF NOMENCLATURE FOR ALGAE, FUNGI, AND PLANTS (Shenzhen Code, Art.
F.8, 2018). However, the application of an asexual name, such as Aecidium,
to a sexual species can cause confusion (Ono 2016). Therefore, we propose
a new combination in Puccinia for A. caulophylli. The holotype specimen
on C. robustum has only spermogonial and aecial stages of the rust; because
the uredinial and telial stages define the rust genus, we designate an epitype
specimen on M. effusum.

Puccinia caulophylli (Kom.) Jing X. Ji & Kakish., comb. nov. Figs 3-5

MB 830631

= Aecidium caulophylli Kom., in Jaczewski & al., Fungi Rossiae Exsicc. 4: no 176, 1898.

Types: Russian Federation, Russian Far East, Siberia, Amur, Mt. Burejenses, stages

0, I on Caulophyllum robustum Maxim. [= C. thalictroides subsp. robustum (Maxim.)

Kitam.], June 1895, leg. V. Komarov (holotype, LE; isotype, NY 00610976). China, Jilin

Province, Yanbian, Erdaobaihe, stages II, III on Milium effusum L., 2 September 2018,

leg. J.X. Ji & M. Kakishima (epitype designated here, HMJAU 8627; MBT 386779).
SPERMOGONIA amphigenous, pale yellow to yellowish brown, subepidermal,
type 4 of Cummins & Hiratsuka (2003). Azcta hypophyllous, yellow,
subepidermal, erumpent, Aecidium-type with firmly connected peridia.
AECIOSPORES Catenulate, subglobose, ovate to ellipsoid, 14-24 x 13.5-20
um (av. 18.5 x 15.5 um), walls hyaline, 0.5-2 um thick (av. 1 um), densely
verrucose.

UreEDINIA mostly hypophyllous, pale yellow to cinnamon-brown,
subepidermal, erumpent, with abundant peripheral and intermixed paraphyses.
PARAPHYSES cylindric to capitate, 16.5-41 x 3-9.5 um (av. 30 x 5 um), walls
hyaline, 0.5-1.5 um thick (av. 1 um). UREDINIOSPORES pedicellate, globose
to subglobose, 22-35 x 21-30.5 um (av. 30 x 27 um), walls hyaline or pale
yellow, 1-3.5 um thick (av. 1.5 um), echinulate, germ pores obscure. TELIA
mostly hypophyllous, dark brown to black, subepidermal, covered by
epidermis, without paraphyses. TELIOSPORES 2-celled by transverse septum,
borne singly on pedicels, clavate to oblong, with round to obtuse apex and
attenuate towards base, 28.5-40.5 x 9.5-17 um (av. 35.5 x 13 um), walls pale
brown to dark brown, 0.5-1.5 um thick at sides (av. 1 um), 1-5 um thick at
apex (av. 2.5 um), smooth; pedicels short, hyaline.

Puccinia caulophylli comb. nov. (China) ... 727

Fic. 4. Puccinia caulophylli on Milium effusum: uredinial and telial stages. A. Uredinia and
telia produced on the leaves; B. Pale yellow uredinia (U) on lower leaf surface; C. Dark brown
telia on lower leaf surface; D. Echinulate urediniospores; E. Vertical section of uredinium with
urediniospores and paraphyses (P); F. Teliospores; G. Vertical section of telia covered by host
epidermis. Scale bars: D, E = 30 um; F, G = 20 um.

728 ... Ji & al.

Fic. 5. Puccinia caulophylli observed by SEM. A. Aecium with catenulate aeciospores and
peridium; B. Aeciospore with densely verrucose surface; C. Uredinium with urediniospores and
paraphyses (P); D. Echinulate urediniospore; E. Vertical section of a telium covered by host
epidermis. Scale bars: A = 30 um; B, D = 5 um; C, E= 20 um.

ADDITIONAL SPECIMENS EXAMINED—Stages 0, I on Caulophyllum robustum:
CHINA: JILIN PROVINCE, Jilin, 23 June 2015 (HMJAU 853); 24 June 2015 (HMJAU
8531, HMJAU 8534); 29 June 2017 (HMJAU 8620); 1 July 2018 (HMJAU 8624).

Stages II, III on Milium effusum: CHINA: JILIN PROVINCE, Jilin, 12 September
2017 (HMJAU 8622); 1 July 2018 (HMJAU 86232). Yanbian, 28 July 2015 (HMJAU
8618).; Baishan, 2 September 2018 (HMJAU 8625); 3 September 2018 (HMJAU 8626).
HEILONGJIANG PROVINCE: Wuchang, 5 July 2016 (HMJAU 8619); 9 September 2017
(HMJAU 8621).

Puccinia caulophylli comb. nov. (China) ... 729

HostTs & DISTRIBUTION—Stages 0, I on Caulophyllum robustum: China (Miura
1928, Tai 1979), Japan (Ito 1950, Harada 1984, Hiratsuka & al. 1992). Stages II,
UI on Milium effusum: China.

Acknowledgments

This work was financed by the Fungal Flora in Jilin Province (20130206073NY).
We thank Dr E.H.C. McKenzie (Manaaki Whenua Landcare Research, Auckland,
New Zealand) and Dr C.M. Denchev (Bulgarian Academy of Sciences, Sofia,
Bulgaria) for critical reading of the manuscript and suggestions. We also thank Dr
H. Koba (College of Arts and Sciences, J. F Oberlin University, Tokyo, Japan) for
identification of M. effusum.

Literature cited

Azbukina ZM. 1984. The manual of rust fungi in Soviet Far East. Nauka, Moscow. (In Russian)

Azbukina ZM. 2005. Rust fungi. Cryptogamic plants, fungi and mosses of the Russian Far East,
vol. 5. Dalnauka, Vladivostok. (In Russian)

Cummins GB. 1971. The rust fungi of cereals, grasses and bamboos. Springer-Verlag, New York.

Cummins GB, Hiratsuka Y. 2003. Illustrated genera of rust fungi, 3 ed. American
Phytopathological Society, St. Paul, Minnesota.

Harada Y. 1984. Materials for the rust flora of Japan IV. Transactions of Mycological Society of
Japan 25: 287-294.

Hiratsuka N, Sato S, Katsuya K, Kakishima M, Hiratsuka Y, Kaneko S, Ono Y, Sato T, Harada
Y, Hiratsuka T, Nakayama K. 1992. The rust flora of Japan. Tsukuba-shuppankai, Tsukuba.

Ito S. 1950. Mycological flora of Japan, vol. 2, no 3. Yokendo, Tokyo.

Ji JX, Li Z, Wan Q, Li Y, Kakishima M. 2016. Notes on rust fungi in China 1. Autoecious
life cycle of Puccinia tatarinovii on Prenanthes. Mycotaxon 131: 653-661.
https://doi.org/10.5248/131.653

Ji JX, Li Z, Wan Q, Li Y, Kakishima M. 2017a. Life cycle of Aecidium klugkistianum on
Ligustrum and its new combination, Puccinia klugkistiana. Mycoscience 58: 307-311.
https://doi.org/10.1016/j.myc.2017.01.004

Ji JX, Li Z, Wan Q, Li Y, Kakishima M. 2017b. Notes on rust fungi in China 3. Puccinia
adenocauli comb. nov. and its life cycle and new host. Mycotaxon 132: 141-148.
https://doi.org/10.5248/132.141

Ji JX, Li Z, Li Y, Kakishima M. 2019. Two new species of Pucciniastrum producing
dimorphic sori and spores from northeast of China. Mycological Progress 18: 529-540.
https://doi.org/10.1007/s11557-018-1460-z

Liu M, Hambleton S. 2013. Laying the formation for a taxonomic review of
Puccinia coronata s.l. in a phylogenetic context. Mycological Progress 12: 63-89.
https://doi.org/10.1007/s11557-012-0814-1

Miura M. 1928. Flora of Manchuria and east Mongolia 3. Cryptogams, Fungi.
Minamimanshutetsudo, Dalian.

Ono Y. 2016. Phakopsora hornotina, an additional autoecious rust species on Meliosma
in the Philippines and the Ryukyu Islands, Japan. Mycoscience 57: 71-78.
https://doi.org/10.1016/j.myc.2015.09.003.

Padamsee M, McKenzie EHC. 2017. The intriguing and convoluted life of a heteroecious rust
fungus in New Zealand. Plant Pathology 66: 1248-1257. https://doi.org/10.1111/ppa.12672

730 ... Ji & al.

Saccardo PA, Sydow P. 1902. Supplementum universale, pars 5. Sylloge fungorum 16. 1291 p.

Scholler M, Lutz M, Aime MC. 2019. Repeated formation of correlated species in Tranzschelia
(Pucciniales). Mycological Progress 18: 295-303. https://doi.org/10.1007/s11557-018-1417-2

Tai FL. 1979. Sylloge fungorum sinicorum. Science Press, Beijing.

Zhuang JY, Wei SX, Wang YC. 1998. Flora fungorum sinicorum, vol.10, Uredinales (1). Science

Press, Beijing.

MY COTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. ©2019

October-December 2019—Volume 134, pp. 731-735
https://doi.org/10.5248/134.731

Exserticlava aquatica sp. nov.,
a microfungus from the Brazilian Amazon

LUANA TEIXEIRA DO CARMO’, DIOGO CARELI DOS SANTOS?,
CAROLINA RIBEIRO SILVA”, SHEILA MIRANDA LEAO FERREIRA’,
THAMARA ARAO FELETTI’, Luis FERNANDO PASCHOLATI GUSMAO”?

' Universidade Estadual de Feira de Santana, Programa de Pés-graduagao em Botanica,
Av. Transnordestina s/n, Novo Horizonte, 44036-900, Feira de Santana, Brazil

? Universidade Federal de Pernambuco, Centro de Biociéncias, Depto de Micologia,
Av. Prof. Nelson Chaves, s/n, Cidade Universitaria, 50670, Recife, Pernambuco, Brazil

* Correspondence to: lgusmao@uefs.br

ABSTRACT—A new species, Exserticlava aquatica, collected on submerged decaying twigs
in Para State in the Brazilian Amazon, is described and illustrated. The microfungus is
characterized by monoblastic conidiogenesis with repeated percurrent conidiophore
extensions and conidiogenous cells with a slightly swollen apex that does not protrude
beyond the ruptured outer wall.

Key worps—Ascomycota, Chaetosphaeriaceae, conidial fungi, freshwater fungi, taxonomy

Introduction

Exserticlava S. Hughes is characterized by distoseptate conidia and a hyaline
apical swelling of the conidiogenous cell formed by the inner wall disrupting a
pigmented outer wall (Hughes 1978). Tsui & al. (2001) reviewed the morphology
of the genus and provided a key to five species; subsequently two more species
were described: E. yunnanensis and E. manglietiae (Cai & Hyde 2007, Ren & al.
2012). Exserticlava is associated with the perithecial genus Chaetosphaeria Tul.
& C. Tul. (Reéblova & Seifert 2003, Seifert & al. 2011: 204). During a survey of
microfungi associated with twigs in estuary areas, an interesting specimen was
collected and is described here as a new Exserticlava species.

732 ... Carmo & al.

Material & methods

Samples of submerged decaying twigs were washed in river water to remove
sand and placed in plastic bags. In the laboratory, the samples were processed
according Castafeda-Ruiz & al. (2016) and regularly examined for 40 days under
stereomicroscope for observation of the reproductive structures. Slide mounts
were prepared in PVL (polyvinyl alcohol, lactic acid, and phenol) and/or in
lactic acid. Microphotographs were obtained using a Olympus BX51 microscope
with Nomarski interference optics. The type specimen has been deposited in the
Herbarium of Universidade Estadual de Feira de Santana, Brazil (HUEFS).

Taxonomy

Exserticlava aquatica L.T. Carmo, C.R. Silva, Careli, S.M. Ledo, Feletti & Gusmao,
sp. nov. PLATE 1
MB 831391
Differs from Exserticlava triseptata by its monoblastic conidiogenesis, its conidiogenous
cells with a slightly swollen apex that does not protrude beyond the ruptured outer wall,
and its smaller oblong-obtuse conidia.

Type: Brazil, Parad State: Belém, Mosqueiro island, 1°03’45”S 48°20'14”W, on
submerged decaying twigs of unidentified plant, 2.V1.2018, coll. L.T. Carmo (Holotype,
HUEFS249946).

EryMo.oey: the specific epithet refers to the aquatic habitat from which the species

was collected.
CoLonigs effuse, brown. MyceLium superficial and immersed, composed
of septate, branched, smooth, pale brown hyphae. CoNnrIDIOPHORES
macronematous, mononematous, unbranched, erect, straight or slightly
flexuous, smooth, thick-walled, dark brown to paler toward the apex, slightly
swollen at the base, surrounded by pseudo-parenchymatous cells forming a
brown stroma 22.5-27 um wide, (6-)8-13-septate, 330-455 x 6.5-7.5 um.
CONIDIOGENOUS CELLS monoblastic, enteroblastic, percurrent and progressive
leading to the production of up to 7 conidiogenous cell extensions, integrated,
terminal, clavate, slightly swollen at the apex, without conspicuous expansion
of the inner wall and not protruding from the fragmented and pigmented outer
wall of conidiogenous cells, smooth, pale brown, 18.5-46.5 x 5-7.5 um. Conidial
secession schizolytic. Conip1a acrogenous, holoblastic, solitary, smooth,
oblong-obtuse, thick-walled, 3-distoseptate, sometimes with conspicuous

PuaTE 1. Exserticlava aquatica (holotype, HUEFS 249946): A. General aspect of conidiophore
with attached conidium; B. Conidiogenous cell development; C. Conidium attached to
conidiogenous cell; D. Conidiogenous cell; E. Proliferating conidiogenous cells; F. Conidiogenous
cell producing a conidium after extension; G-I. Conidia. Scale bars: A = 50 um; B-I = 10um.

Exserticlava aquatica sp. nov. (Brazil) ... 733

734 ... Carmo & al.

aperture on distosepta, pale brown, 21-26.5 x 10-11.5 um; conidia sometimes
produced within intercalary old conidiogenous cells. SEXUAL MORPH: not
observed.

Note: Exserticlava triseptata, E. keniensis, and E. vasiformis most closely
resemble E. aquatica by producing 3-distoseptate conidia; the four other
accepted species are 1-distoseptate (Tsui & al. 2001, Rao & de Hoog 1986).
However, E. triseptata and E. keniensis have brown elliptic-obovoid conidia
and the conidiogenous cells extend forming a hyaline swelling that produces
conidia; and E. vasiformis is distinguished by vigorous inner wall extension of
funnel-shaped conidiogenous loci (Hughes 1978, Matsushima 1975, Tsui &
al. 2001). Exserticlava aquatica is easily separated from the other Exserticlava
species by its pale brown conidia, its several percurrent conidiogenous cell
extensions, and the distinctive crown-like appearance of the outer wall of the
conidiogenous cell; the new species is further characterized by monoblastic
conidiogenous cells and an absence of conspicuous inner wall expansion to
fragment the pigmented outer conidiogenous cell wall.

Exserticlava triseptata and E. vasiformis have been widely recorded in
Brazil, including in the Amazon region (Cruz & al. 2008).

Acknowledgments

We are indebted to Dr. De-Wei Li and Dr. Rafael F. Castafieda Ruiz for critical
review of the manuscript. LTC and TAF thank the Programa de Pds-graduacao em
Botanica (PPGBot/UEFS), the Nacional Council for Scientific and Technological
Development (CNPq), and the “Coordenacgao de Aperfeigoamento de Pessoal de
Nivel Superior (CAPES). DCS and CRS thank the Programa de Pés-graduacao
em Biologia de Fungos (PPGBF/UFPE). LFPG is grateful to the CNPq (Proc.
312984/2018-9).

Literature cited

Cai L, Hyde KD. 2007. Anamorphic fungi from freshwater habitats in China: Dictyosporium
tetrasporum and Exserticlava yunnanensis spp. nov. and two new records for
Pseudofuscophialis lignicola and Pseudobotrytis terrestris. Mycoscience 48(5): 290-296.
https://doi.org/10.1007/s10267-007-0369-1

Castafieda-Ruiz RF, Heredia G, Gusmao LFP, Li DW. 2016. Fungal diversity of Central and
South America. 197-217, in: DW Li (ed.). Biology of microfungi... New York, Springer
International Publishing. https://doi.org/10.1007/978-3-319-29137-6_9

Cruz ACR, Hernandez-Gutiérrez A, Gusmao LFP. 2008. O género Exserticlava (fungo
anamorfo—Hyphomycetes) no Brasil. Revista Brasileira de Botanica 31(2): 357-361.
https://doi.org/10.1590/S0100-84042008000200018.

Hughes SJ. 1978. New Zealand fungi 25. Miscellaneous species. New Zealand Journal of
Botany16(3): 311-370. https://doi.org/10.1080/0028825X.1978.10425143

Exserticlava aquatica sp. nov. (Brazil) ... 735

Matsushima T. 1975. Icones microfungorum a Matsushima lectorum. Kobe, Published by the
author. 209 p.

Rao V, de Hoog GS. 1986. New or critical hyphomycetes from India. Studies in Mycology 28.
84 p.

Réblova M, Seifert KA. 2003. Six new species of Chaetosphaeria from tropical rain forests in
Thailand and redescription of Chaetosphaeria hiugensis. Sydowia 55: 313-347.

Ren SC, MaJ, Zhang XG. 2012 [“2011”]. Two new species of Exserticlava and Spiropes on decaying
wood from Guangdong, China. Mycotaxon118: 349-353. https://doi.org/10.5248/118.349

Seifert K, Morgan-Jones G, Gams W, Kendrick B. 2011. The genera of hyphomycetes. CBS
Biodiversity Series 9. 997 p.

Tsui CK, Goh TK, Hyde KD. 2001. A revision of the genus Exserticlava, with a new species.
Fungal Diversity 7: 135-143.

MYCOTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. © 2019

October-December 2019—Volume 134, pp. 737
https://doi.org/10.5248/134.737

Regional annotated mycobiotas new to the Mycotaxon website

ABSTRACT—Mycotaxon is pleased to add to our ‘web-list’ page the following new annotated
species distribution list under South America (Brazil): “Ascomycota (lichenized and non-
lichenized) on Syagrus coronata in the Caatinga biome: new and interesting records for
Brazil and South America” by Maiara A.L. dos Santos, Nilo G.S. Fortes, Tassio E.F. Silva,
Nadja S. Vitoria. This brings to 133 the number of free access Fungae now available on our
website: http://www.mycotaxon.com/mycobiota/index.html

SOUTH AMERICA
Brazil

Matra A.L. DOs SANTOS, NILO G.S. FoRTES, TAssio E.F. Sitva, NADJA S. VITORIA.

Ascomycota (lichenized and non-lichenized) on Syagrus coronata in the
Caatinga biome: new and interesting records for Brazil and South America.
10 p.
ABSTRACT—The Caatinga biome occupies most of the semiarid region of
northeastern Brazil, with varied landscapes and notable endemism. Among the
plants having significant importance in the Caatinga environment is the palm tree
Syagrus coronata, which is known as the “life-saving plant” due to its high socio-
biological and economic value. To better understand the mycota of the Arecaceae,
collections were undertaken in the municipalities of Paulo Afonso and Nova Gloria
within the Raso da Catarina eco-region in the drylands (“sertao”) of Bahia State,
Brazil. Twenty species of Ascomycota were identified during the present work:
three are new records for South America (Diplodia galiicola, Seimatosporium
corni, and Wojnowiciella viburni); eleven are new records for Brazil (Anthostomella
caricis, Caryospora callicarpa, C. putaminum, Chaetomium subaffine, Diatrype
bermudensis, Diatrypella persicae, Didymosphaeria massarioides, Eutypella
fraxinicola, Munkovalsaria donacina, Oedohysterium sinense, and Pleospora
calvescens); while six are new records for Bahia State (Dirinaria confusa, Lecanora
achroa, Phaeosphaeria sp., Pleospora herbarum, Polymeridium julelloides, and
Saccardoella macrasca). Syagrus coronata represents a new botanical host for all
taxa identified here.

Key worps—Pezizomycotina, semiarid, taxonomy

MYCOTAXON

ISSN (print) 0093-4666 (online) 2154-8889 Mycotaxon, Ltd. © 2019

October-December 2019—Volume 134, pp. 739
https://doi.org/10.5248/134.739

Regional annotated mycobiotas new to the Mycotaxon website

ABSTRACT—MycotTaxon is pleased to add a new annotated species distribution list
to our 134 previously posted free access fungae. The 22-page “Checklist of Bolivian
Agaricales. 1: Species with dark and pink spore prints.” by E. Melgarejo-Estrada, M.E.
Suarez, D. Rocabado, O. Maillard, and B.E. Lechner may be downloaded from our
website via http://www.mycotaxon.com/mycobiota/index.html

SOUTH AMERICA
Bolivia

E. MELGAREJO-EsTRADA, M.E. SUAREZ, D. ROCABADO, O. MAILLARD, B.E. LECHNER.
Checklist of Bolivian Agaricales. 1: Species with dark and pink spore prints.
22 p.
ABSTRACT—We provide a literature-based checklist of Agaricales reported from
Bolivia. In this first contribution, 101 species belonging to 28 genera and 9 families
are listed. Pluteaceae, Agaricaceae and Hymenogastraceae are the most species-
abundant families.

Key worps—Basidiomycota, distribution, diversity, Gasteromycetes, Neotropics,
macromycetes, South America

Exserticlava aquatica sp. nov.
(Carmo & al.— Pate 1, p. 733)